Methods For Selectively Modulating Survivin Apoptosis Pathways

ABSTRACT

The present invention, based on the discovery of a new biological phenomena, provides methods and compositions for use in identifying agents that modulate the phosphorylation of survivin, the interaction between survivin and p34 cdc2 -cyclin B1 kinase complex, and the interaction between survivin and caspase-9. Related methods and compositions can be used to modulate survivin regulated apoptosis.

RELATED APPLICATIONS

This application is related to provisional application Ser. No.60/080,288, filed Apr. 1, 1998, International ApplicationPCT/US99/07205, filed Apr. 1, 1999, application Ser. No. 09/283,144,filed Apr. 1, 1999, provisional application Ser. No. 60/031,435, filedNov. 20, 1996, application Ser. No. 08/975,080, filed Nov. 20, 1997 andInternational Application PCT/US97/21880, filed Nov. 20, 1997, all ofwhich are herein incorporated by reference in their entirety.

ACKNOWLEDGMENT OF FEDERAL SUPPORT

The research and discoveries described herein were supported by grantsHL 43773 and HL 54131 from the National Institutes of Health.

TECHNICAL FIELD

The present invention, based on the discovery of a new biologicalphenomena, provides methods and compositions for use in identifyingagents that modulate the phosphorylation of survivin, the interactionbetween survivin and p34^(cdc2)cyclin B1 kinase complex, and theinteraction between survivin and caspase-9. Related methods andcompositions can be used to modulate survivin regulated apoptosis.

BACKGROUND OF THE INVENTION

A. The Role of Survivin in Programmed Cell Death

Programmed cell death (sometimes referred to as apoptosis) isdistinguishable, both morphologically and functionally, from necrosis.Programmed cell death is a natural form of death that organisms use todispose of cells. Cells dying by programmed cell death usually shrink,rarely lyse, and are efficiently removed from the organism (rapidlyrecognized and engulfed by macrophages) without the appearance ofinflammation (Michael Hengartner, “Cell Death and Aging, MolecularMechanisms of,” IN MOLECULAR BIOLOGY AND BIOTECHNOLOGY 158-62 (ed. R. A.Meyers, 1995)).

Apoptosis was initially used to describe a subset of programmed celldeaths sharing a particular set of morphological features which includemembrane blebbing, shrinkage of cytoplasm, chromatic condensation andformation of a “DNA ladder.” During apoptosis, cells lose their celljunctions and microvilli, the cytoplasm condenses and nuclear chromatinmarginates into a number of discrete masses. While the nucleusfragments, the cytoplasm contracts and mitochondria and ribosomes becomedensely compacted. After dilation of the endoplasmic reticulum and itsfusion with the plasma membrane, the cell breaks up into severalmembrane bound vesicles, referred to as apoptotic bodies, which areusually phagocytosed by adjacent cells. Activation of particular genessuch as tumor suppressor genes in vertebrates is thought to be necessaryfor apoptosis to occur. Apoptosis induced by numerous cytotoxic agentscan be suppressed by expression of the gene bcl-2, which produces acytoplasmic protein Bcl-2 (THE ENCYCLOPEDIA OF MOLECULAR BIOLOGY 67 (ed.John Kendrew et al., Blackwell Science; Oxford, England, 1994).

Survivin has recently been identified as a novel inhibitor of apoptosis(IAP). The gene encoding survivin is located on chromosome 17q25.Survivin is a 16.5 kD cytoplasmic protein containing a single partiallyconserved BIR domain, and a highly charged carboxyl-terminus coiled-coilregion instead of a RING finger, which inhibits apoptosis induced bygrowth factor (IL-3) withdrawal when transferred in B cell precursors(Ambrosini, G. et al., 1997). Unlike other members of the IAP survivinhas only one BIR domain and does not have a carboxy-terminal RINGfinger. Instead, survivin has a carboxy-terminus coiled-coil region.Based on overall sequence conservation, the absence of a carboxyterminus RING finger and the presence of a single, partially conserved,BIR domain, survivin is the most distantly related member of the IAPfamily, sharing the highest degree of similarity with NAIP (Roy, N. etal., 1995). Additionally, unlike other IAP proteins, survivin isundetectable in adult tissues, but becomes prominently expressed in allthe most common human cancers of lung, colon, breast, pancreas, andprostate, and in ˜50% of high-grade non-Hodgkin's lymphomas, in vivo.

Expression of survivin in embryonic and fetal development may contributeto tissue homeostasis and differentiation that is independent of bcl-2(Adida et al., 1998). Aberrations of this survivin-associateddevelopmental pathway may result in prominent re-expression of survivinin neoplasia and abnormally prolonged cell viability (Adida et al.,1998).

Deregulation of programmed cell death has emerged as a primary mechanismcontributing to the pathogenesis of various human diseases includingcancer. While the impact of anti-apoptosis genes in neoplasia hasfocused, for example, on the role of bcl-2 in follicular lymphoma, apotential distribution of IAP proteins, such as survivin, has only begunto been investigated. Survivin is rarely present in adult tissues buthas been detected in adenocarcinoma of the pancreas, breastadenocarcinoma, colon cancer, head and neck squamous cell carcinoma,neuroblastoma, malignant thymoma, prostate cancer, and benign prostatehyperplasia (see U.S. Ser. No. 08/975,080). This expression patternsuggests that overexpression of survivin or alterations in survivin generegulation may commonly occur during tumorigenesis.

B. Cell Cycle

Living organisms are composed of cells, whose growth and divisionrequire a regular sequence of events and processes that comprise thecell cycle. Some cell cycle events are continuous (e.g., synthesis ofproteins and lipids), whereas others are discontinuous (e.g., DNAsynthesis). Two discontinuous processes for cell survival are thereplication of DNA and the segregation of chromosomes to the daughtersof cell division during mitosis. If either of these steps are performedinaccurately, the daughter cells will be different from each other andwill almost certainly be flawed. Segregation of chromosomes occursduring mitosis, normally a relatively brief period in the cell cycle,which culminates in the highly visible act of cell division (e.g.,cytokinesis). The remainder of cell cycle comprises interphase, duringwhich growth occurs. Chromosome replication occurs in eukaryotic cellsonly during interphase; and replication and segregation are mutuallyexclusive processes.

Interphase is subdivided into the S phase when DNA synthesis occurs andthe gaps separating S phase from mitosis. G1 is the gap after mitosis,before DNA synthesis starts; G2 is the gap after DNA synthesis iscomplete, before mitosis and cell division. Cellular constituents directthe cell cycle by acting as regulatory elements.

C. Checkpoint Mechanisms for Apoptosis and Cell Cycle

One of the central functions of apoptosis (programmed cell death) inmaintaining homeostasis is the elimination of damaged and potentiallyharmful cells (Vaux and Korsmeyer, 1999). For this process to beeffective, the apoptotic machinery must constantly couple tosurveillance mechanisms, i.e. “checkpoints”, sensing DNA damage, adverseenvironmental conditions, and oncogene or viral transformation (Hunter,1997; Paulowich et al., 1997). Checkpoint activation under theseconditions initiates apoptosis (Evan and Littlewood, 1998) via theassembly of an evolutionary conserved “apoptosome”, which in mammaliancells comprises an upstream cell death protease, caspase-9, theadapter/cofactor protein Apaf-1, mitochondria-derived cytochrome c anddATP/ATP (Green, 1998). Although it is debated how apoptosome assemblypromotes caspase-9 catalytic activity (Rodriguez and Lazebnik, 1999; Zouet al., 1999), this process culminates with downstream activation ofeffector caspases and cleavage of critical cellular substrates (Salvesenand Dixit, 1997; Thornberry and Lazebnik, 1998). A similar paradigmlinking apoptosis control to checkpoint activation (Levine, 1997), hasbeen extended to surveillance mechanisms presiding over cell cycletransitions (Pines, 1999), assembly of a bipolar mitotic apparatus(Merdes and Cleveland, 1997), preservation of ploidy (Nicklas, 1997),and timing of cytokinesis (Field et al., 1999). In this context,dysregulated expression of apoptosis inhibitors bcl-2 and bcl-XL hasbeen shown to restrain S phase entry (Linette et al., 1996), promotecell cycle exit (Huang et al., 1997), and cause aneuploidy (Mimi et al.,1996), further demonstrating a role of the apoptotic machinery in cellcycle progression.

Survivin is expressed in G2/M in a cell cycle-dependent manner, andlocalized to mitotic spindle microtubules and intercellular acto-myosinbridges, i.e. midbodies, during cell division (Li et al., 1998).Interference with this topography, or blocking survivin expression,caused increased caspase-3 activity in G2/M (Li et al., 1998), and aprofound dysregulation of mitotic progression (Li et al., 1999),suggesting that survivin may regulate a novel apoptotic checkpoint atcell division. This pathway was dramatically exploited in cancer(Ambrosini et al., 1997), where survivin was identified as one of thetop four “transcriptomes” out of 3.5 millions mRNAs, uniformly expressedin cancer, but not in normal tissues (Velculescu et al., 1999).Additionally, it has been shown that transformed cells are exquisitelysensitive to manipulation at this mitotic checkpoint as interferencewith survivin expression and function using dominant-negative mutantswith point mutations in the conserved baculovirus IAP repeat (BIR)domain or survivin antisense resulted in aberrant mitoses (Li et al.,1999) and spontaneous apoptosis (Ambrosini et al., 1999; Grossman etal., 1999a; Grossman et al., 1999b). This phenotype is unique tosurvivin and not observed with other apoptosis inhibitors potentiallycontributing to neoplasia, as antisense inhibition of Bcl-2 increasedsensitivity to apoptosis but did not in itself induce cell death (Jansenet al. 1998).

The present invention identifies a mechanism by which survivin mayintegrate the control of cell division with the regulation of apoptosisin mammalian cells. The present invention also provides two classes ofsurvivin antagonists that modulate the expression of survivin andinterfere with the antiapoptotic survivin pathway in melanoma tumors invivo. Further, the present invention provides a method of inhibiting thegrowth of tumor comprising administering an effective amount of asurvivin antagonist to the tumor.

SUMMARY OF THE INVENTION

The present invention is based in part on the finding that survivin isphosphorylated by the main mitotic kinase complex, p34^(cdc2)-cyclin B1(Nurse, 1994), and that this process is essential to preserve viabilityof cells traversing mitosis.

The present invention is also based in part on the finding that lack ofsurvivin phosphorylation by p34^(cdc2) causes dissociation of thesurvivin-active caspase-9 complex, selective mislocalization ofcaspase-9 from midbodies, and caspase-9-dependent apoptosis of cellstraversing mitosis.

The present invention provides a method of identifying an agent thatmodulates the phosphorylation of survivin comprising the steps ofincubating survivin and p34^(cdc2)-cyclin B1 kinase complex with anagent and determining whether the agent modulates the phosphorylation ofsurvivin, thereby identifying an agent that modulates phosphorylation ofsurvivin.

The present invention further provides methods of identifying an agentthat modulates the interaction of survivin and p34^(cdc2)-cyclin B1kinase complex.

The invention also provides methods of modulating the interactionbetween survivin and p34^(cdc2)-cyclin B1 kinase complex comprising thestep of administering an effective amount of an agent which modulates atleast one interaction between Survivin and p34^(cdc2)-cyclin 81 kinasecomplex.

The invention further includes methods of modulating apoptosis in acell, comprising administering to the cell an effective amount of anagent that modulates the interaction between survivin andp34^(cdc2)-cyclin B1 kinase complex. The invention further providesagents, compositions, and peptides that modulate the interactionsbetween Survivin and p34^(cdc2)-cyclin B1 kinase complex.

The present invention also provides a method of identifying agents thatmodulate the interaction between phosphorylated survivin and caspase-9.The invention further provides agents, compositions, and peptides thatmodulate the interactions between phosphorylated survivin and caspase-9.

The present invention provides survivin antagonists such as the dominantnegative survivin mutant (Thr³⁴→Ala) and survivin antisense nucleicacid, to modulate the expression of survivin in a cell. The inventionalso discloses using the survivin antagonists to inhibit the growth oftumors in vivo and in vitro.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D. p34^(cdc2)Cyclin B1 Phosphorylation of Survivin on Thr³⁴.

FIG. 1A. Clustal alignment of survivin and IAP proteins. A consensusp34^(cdc2) phosphorylation sequence (S/TPXR/K) surrounding Thr³⁴ insurvivin is boxed.

FIG. 1B. In vitro kinase assay. Wild type (WT) survivin, survivin (T34A)(T34A), or control (H1) were incubated with baculovirus-expressedp34^(cdc2)-cyclin B1 or cdk2-cyclin E in kinase buffer plus 10 μCiγ-³²P-ATP for 30 min at 30° C. Samples were separated by SDS gelelectrophoresis and radioactive bands were visualized byautoradiography. Equal protein loading was confirmed by Coomassie bluestaining of the gel.

FIG. 1C. Reactivity with an antibody to phosphorylated survivin Thr³⁴.The experimental conditions for kinase assays are the same as in B,except that samples were immunoblotted with an antibody raised againstthe survivin peptide sequence L²⁸ EGCACT*PERMAEAGFI⁴⁴ containingphosphorylated Thr³⁴(T*), and sequentially affinity purified onnon-phosphorylated/phosphorylated peptide-Sepharose columns (-phosph.T34), or with an antibody raised against recombinant survivin(-survivin) (Grossman et al., 1999).

FIG. 1D. In vivo phosphorylation of survivin. HeLa cells weretransfected with HA-survivin, labeled with 200 μCi/ml ³² Pi inphosphate-free DMEM medium and immunoprecipitated with control IgG oranti-HA antibody with visualization of radioactive bands byautoradiography. Experiments were repeated at least three times withcomparable results. For all panels, relative molecular weight markersare indicated on the left.

FIGS. 2A-C. Physical Association Between Survivin and p34^(cdc2).

FIG. 2A. Cyclin-dependent kinase immunoprecipitation. HeLa cellsasynchronously growing (Async.) or synchronized to G1, S, or G2/M weredetergent-solubilized and immunoprecipitated with antibodies top34^(cdc2) (left panel), or Cdk2 (right panel). Samples wereelectrophoresed, transferred to nylon 34 membranes and separatelyimmunoblotted with antibodies to survivin, p34^(cdc2) or Cdk2,respectively.

FIG. 2B. Survivin immunoprecipitation. The experimental procedures arethe same as in A, except that asynchronously growing HeLa cells weretransfected with HA-survivin or HA-survivin (T34A), immunoprecipitatedwith an antibody to HA, and immunoblotted with antibodies to p34^(cdc2)or HA. W, whole HeLa cell extract.

FIG. 2C. Co-localization of survivin and p34^(cdc2), in vivo. HeLa cellson optical grade glass coverslips were fixed in acetone/methanol andsimultaneously incubated with mAb 8E2 to survivin and a rabbit antibodyto p34^(cdc2), followed by FITC-conjugated goat anti-mouse (survivin,green) and TR-conjugated goat anti-rabbit (p34^(cdc2), red) antibodies.Images were analyzed by confocal laser-scanning microscopy at theindicated cell cycle phases. Image merging analysis is shown on theright of each panel. For panels A and B, relative molecular weightmarkers are indicated on the left.

FIGS. 3A-D. Expression of Non-Phosphorylatable Survivin (T34A) InducesSpontaneous Apoptosis and Dysregulation of Mitosis.

FIG. 3A. Nuclear morphology. HeLa cells transfected with GFP-vector(vector), GFP-survivin (T34A) (T34A), or GFP-caspase-9 (Met¹-Asp³³⁰)(caspase-9), were scored morphologically for nuclear integrity by DANstaining after 48-h. Arrows, nuclear fragmentation and chromatincondensation in GFP-expressing cells.

FIG. 3B. Summary of HeLa cell apoptosis induced by expression ofsurvivin (T34A). The experimental procedures are the same as in A. Dataare the meavSEM of four independent experiments.

FIG. 3C. Caspase-dependent apoptosis induced by survivin (T34A). HeLacells were transfected with GFP-survivin (T34A) in the presence or inthe absence of 20 μM of the caspase inhibitor, Z-VAD-fmk. GatedGFP-expressing cells were analyzed 48 h after transfection for DNAcontent by propidium iodide staining and flow cytometry. The percentagesof apoptotic cells with sub-G1 (hypodiploid) 35 DNA content areindicated. Apoptotic HeLa cells in GFP-vector transfectants were 8%.Data are representative of one experiment out of at least threeindependent determinations.

FIG. 3D. Aberrant mitoses. HeLa cells transfected with wild typesurvivin (WT) or survivin (T34A) (T34A), were fixed inmicrotubule-stabilizing buffer (Li et al., 1998), and labeled with mAb20C6 to β-tubulin followed by FITC-conjugated goat anti-mouse IgG.Images were analyzed by confocal microscopy as described in FIG. 2C.

FIGS. 4A-C. Apoptosis Induced by Survivin (T34A) Coincides with Mitosis.

FIG. 4A. Tet-regulated YUSAC-2 transfectants. Three YUSAC-2 melanomacell lines stably transfected with survivin (T34A) in pTet splice wereanalyzed for DNA content by propidium iodide staining and flow cytometryin the presence (Tet+) or absence (Tet−) of Tet. The percentages ofcells with sub-G1 (apoptotic) DNA content after a 3-d culture at 37 Care indicated.

FIG. 4B. TUNEL labeling. The experimental conditions are the same as inA, except that a subclone of YUSAC-2 transfectants, F5.C4 was analyzedin the presence (Tet+) or in the absence (Tet−) of Tet forinternucleosomal DNA fragmentation by end-labeling with terminaldeoxynucleotidyl transferase (TdT) and a peroxidase-conjugatedanti-digoxigenin antibody.

FIG. 4C. Cell cycle analysis. F5.C4 cells were synchronized at the G1/Sboundary in the presence (Tet+) or absence (Tet−) of Tet, released, andanalyzed for changes in DNA content at 3-h intervals by propidium iodidestaining and flow cytometry. The percentages of cells with sub-G1(apoptotic), G1, S, or G2/M DNA content are indicated for each timepoint analyzed. Data are representative of one experiment out of atleast three independent determinations.

FIGS. 5A-D. Thr³⁴ Phosphorylation Changes the Specificity ofSurvivin-Caspase-9 Interaction at Mitosis.

FIG. 5A. Phosphorylation-independent association of survivin-proformcaspase-9 in interphase cells. Asynchronously growing HeLa cells weretransfected with HA-wild type survivin (WT) or HA-survivin (T34A)(T34A), detergent-solubilized after 24 h and immunoprecipitated with anantibody to HA followed by Western blotting with an antibody tocaspase-9. Arrow, position of ˜46 kDa proform caspase-9.

FIG. 5B. Specificity of survivin-caspase-9 interaction. The experimentalconditions are the same as in A, except that HA-survivin and HA-survivin(T34A) immunoprecipitates were separately immunoblotted with antibodiesto caspase-8 or HA. S, supernatant of HA-survivin immunoprecipitate.Arrow, position of ˜55 kDa proform caspase-8.

FIG. 5C. Modulation of survivin-caspase-9 interaction in cycling cells.The experimental conditions are the same as in A, except that extractsfrom adherent (A) or non-adherent (N-A), i.e. mitotic/apoptotic, HeLacells were harvested 48-h after transfection. Aliquots of theimmunoprecipitates (pellet, P) or their supernatants (S) wereimmunoblotted with antibodies to caspase-9 or HA. Arrows, position of˜46 kDa proform caspase-9 and ˜35 kDa active caspase-9. W, whole extractfrom mitotic/apoptotic HeLa cells.

FIG. 5D. Phosphorylation-dependent modulation of survivin-activecaspase-9 recognition at mitosis. HeLa cells were transfected with theindicated HA-constructs, synchronized to the G1/S boundary by a 16-htreatment with thymidine and released. Aliquots of the various cultureswere immunoprecipitated at the indicated time intervals with an antibodyto HA, and immunoblotted with antibodies to caspase-9 or HA. P, pellet;S, supernatant. For all panels, molecular weight markers are indicatedon the left. For all panels, experiments were repeated at least twicewith comparable results.

FIGS. 6A-C. Expression of Survivin (T34A) Mislocalizes Caspase-9 fromMidbodies at Telophase.

HeLa cells transfected with HA-wild type survivin (Survivin) (A),survivin (T34A) (B), or survivin (L64A) (C) were labeled with a mAb toHA (A, B) or mAb 8E2 (C) and a rabbit antibody to caspase-9. Binding ofthe primary antibodies was detected by addition of FITC-conjugated goatanti-mouse (survivin, green) or TR-conjugated anti-rabbit (caspase-9,red) antibody, and analyzed by confocal microscopy image merginganalysis of the individual staining is shown on the right of each panel.Arrows, localization of wild type/mutant survivin and caspase-9 atmidbodies at telophase. Experiments were repeated at least four timeswith comparable results.

FIGS. 7A-B. Caspase-9 Dependent Apoptotic Checkpoint at Cell Division.

FIG. 7A. Proteolytic activation of caspase-9 at mitosis. Tet+ andTet-F5.C4 cells characterized as described in FIG. 4, were harvested atthe indicated time intervals after thymidine release and immunoblottedwith an antibody to caspase-9. TNF+CHX, extract of HeLa cells treatedwith 10 ng/ml TNFα plus 10 μg/ml cycloheximide. Arrows, position of ˜46kDa proform caspase-9, and ˜35 kDa and ˜37 kDa active caspase-9 bands.

FIG. 7B. Effect of caspase-9 (C287A) dominant negative mutant onapoptosis induced by survivin (T34A). HeLa cells were transfected withthe various indicated combinations of GFP-constructs, with or withoutetoposide (10 μg/ml) or TNFα (10 ng/ml) plus cycloheximide (10 μg/ml),harvested after 48 h, and GFP-labeled cells were morphologically scoredfor nuclear fragmentation by DAPI staining, as described in FIG. 2B. DN,dominant negative. Data are the mean±SEM of four independentexperiments.

FIGS. 8A-D. Tet-regulated induction of survivin BIR mutant, apoptosis inF5.C4 cells, and tumor formation in CB.17 mice.

FIG. 8A. F5.C4 cells were cultured in the presence or absence of tet(0.5 μg/ml) and harvested at the indicated time intervals. Lysates weresubjected to Western blotting with antibodies to survivin (top panel) orβ-actin (bottom panel) as described (Grossman et al., 1999). Given themassive expression of the BIR mutant, autoradiography exposure time waslimited in order to visualize discreet bands. The endogenous wild-typesurvivin protein in uninduced (+tet) cells is not apparent at this levelof exposure, but was apparent at longer exposure times.

FIG. 8B. F5.C4 cells were cultured in the presence or absence of tet forthe indicated time intervals, and both non-adherent and adherent cellswere recovered. Cells were then fixed, permeabilized and stained withpropidium iodide for DNA content as described (Grossman et al., 1999).The marker indicates the sub-G₁ fraction, corresponding to apoptoticcells.

FIG. 8C. F5.C4 cells, were plated on coverslips treated with 2% gelatin(Sigma), and incubated 48 hours in the presence or absence of tet. Cellswere fixed in 1% paraformaldehyde for 10 minutes at room temperature andthen permeabilized with acetic acid and ethanol for 5 minutes at −200°C. Arrows indicate TUNEL-reactive (Shockett et al., 1995) cells withapoptotic morphology.

FIG. 8D. YUSAC-2 parental cells (3×10⁶, top panel) or F5.C4 cellstransfected with the survivin Thr³⁴→Ala mutant (bottom panel) wereinjected subcutaneously into CB.17 mice, and tet (100 μg/ml) was added(left side of panels) or withheld (right side of panels) from thedrinking water as indicated. Photographs were taken of representative 8weeks following injection.

FIGS. 9A-B. In Vitro Characterization of Survivin Antisense-TransfectedSubclone B8.

FIG. 9A, DNA content analysis, performed as described in FIG. 1.

FIG. 9B. RT-PCR analysis of cells cultured in the presence or absence oftet as indicated. RNA prepared using TriReagent, and reversetranscription primed with a survivin sense primer.

FIGS. 10A-B. Effect of Survivin BIR Mutant Expression in EstablishedTumors In Vivo, and Tet-Regulated Apoptosis of Tumor LinesRe-Established In Vitro.

FIG. 10A. Growth of F5.C4 tumors in 5 animals maintained on tet (closedtriangles). Error bars indicate standard deviation. Also shown are 5representative growth curves from 15 animals in which tet was withheld(day 0) upon tumor formation (no symbols).

FIG. 10B, A representative F5.C4 tumor following removal of tet wasexpanded in vitro in the presence of G418 and tet (27), and then afterculturing for 72 hours in the presence (top panel) or absence (bottompanel) of tet was examined for DNA content as in FIG. 1.

FIGS. 11A-B. Histologic Analysis of YUSAC-2 Tumor.

Tumor formed by untransfected YUSAC-2 cells was stained with (A)hematoxylin and eosin and (B) HMB-45 antibody.

FIG. 12. Tumor Formation in CB.17 Mice with F5.E5 Cells.

F5.E5 cells (3×10 6, top panel) or 138 cells (bottom panel) wereinjected subcutaneously into CB.17 mice, and tet (100 mg/ml) was added(left side of panels) or withheld (right side of panels) from thedrinking water.

FIG. 13. Summary of the Role of Survivin During Cell Cycle Progression.

A schematic diagram disclosing the role of survivin during cell cycleprogression.

DETAILED DESCRIPTION OF THE INVENTION I. General Description

The present invention is based in part on the finding that survivin isphosphorylated by the main mitotic kinase complex, p34^(cdc2)-cyclin B1(Nurse, 1994), and that this process is essential to preserve viabilityof cells traversing mitosis.

The present invention is also based in part on the finding that survivinbinds to p34^(cdc2)-cyclin B1 kinase complex. Moreover, the presentinvention is based in part on the finding that phosphorylated survivininteracts with caspase-9.

The phosphorylation of survivin protein by p34^(cdc2)-cyclin B1 kinasecomplex, the binding of survivin to the kinase complex, and the bindingof survivin to caspase-9 can be used to identify agents, or serve as atarget for agents, that inhibit or stimulate survivin mediatedfunctions. The agents may be used to modulate survivin mediatedinhibition of cellular apoptosis, to block abnormal cell growth or toextend cell growth in culture. As used herein, modulation of apoptosismeans increasing or decreasing the number of cells that would otherwiseundergo apoptosis in a given cell population. This can be effected bymodulating (increasing or decreasing) phosphorylation of survivin byp34^(cdc2)-cyclin B1 kinase complex, the binding of survivin to thekinase complex, or the binding of phosphorylated survivin to caspase-9.Preferably, the given cell population in which apoptosis is to bemodulated is found in a tumor or other tissue or group of cells in whichbeneficial effects result from the modulation. Preferably, the increaseor decrease in number of cells that would otherwise undergo apoptosis ina given cell population is at least about 10%, 20%, 40% or morepreferably at least about 50% of the cells in that population.

Additionally, the present invention is based in part on the finding thatsurvivin antagonists such as dominant negative survivin mutant(Thr³⁴→Ala) and antisense survivin nucleic acids inhibit the expressionof endogenous survivin in a melanoma and inhibits the growth of themelanoma in vivo and in vitro. Accordingly, the present invention isalso based in part on the finding that administering an effective amountof a survivin antagonist to tumors inhibits the growth of tumors.

II. Specific Embodiments

A. Methods to Identify Agents that Modulate or Block Phosphorylation ofSurvivin by, Binding of Survivin to p34^(cdc2)-Cyclin B1 Kinase Complex,or Binding of Survivin to Caspase-9

As set forth above, the present invention provides method foridentifying agents that modulate, reduce, or block the phosphorylationof survivin by p34^(cdc2)-cyclin B1 kinase complex. The presentinvention also provides methods for identifying agents that, modulate,reduce or block the association of survivin with p34^(cdc2)-cyclin B1kinase complex. The present invention also provides methods foridentifying agents that modulate reduce or block the association ofsurvivin to caspase-9.

In one assay format, survivin is mixed with p34^(cdc2)-cyclin B1 kinasecomplex in the presence and absence of an agent to be tested. Aftermixing under conditions that allow association of survivin with thekinase complex, the two mixtures are analyzed and compared to determineif the agent modulated, increased, promoted, reduced or blocked thephosphorylation of survivin or the binding of survivin to thep34^(cdc2)-cyclin B1 kinase complex. Likewise in the assay forphosphorylated survivin/caspase-9 or unphosphorylated survivin/caspase-9(See Example 3), phosphorylated survivin is mixed with caspase-9 orunphosphorylated survivin is mixed with either proform/active caspase-9of ˜46 kDa and ˜35 kDa, respectively, in the presence and absence of atest agent to determine if the agent modulated, increased, promoted,reduced or blocked the interaction of the two proteins.

Agents that block or reduce the association of survivin with thep34^(cdc2)-cyclin B1 kinase complex or association of survivin andcaspase-9 may be identified by their ability to decrease the amount ofassociation present in the sample containing the test agent. Agents thatblock or reduce the phosphorylation of survivin by p34^(cdc2)-cyclin B1kinase complex may be identified by their ability to decrease the amountof survivin in the sample containing the test agent.

As used herein, an agent is said to reduce or block the phosphorylationof survivin by p34^(cdc2)-cyclin B1 kinase complex when the presence ofthe agent decreases the amount of survivin or totally blocks thephosphorylation of survivin. One class of agents will reduce or blockthe phosphorylation of survivin by binding to the p34^(cdc2)-cyclin B1kinase complex while another class of agents will reduce or block thephosphorylation of survivin by binding to survivin.

As used herein, an agent is said to reduce or blocksurvivin/p34^(cdc2)-cyclin B1 kinase complex association when thepresence of the agent decreases the extent to which or prevents thekinase complex from becoming associated with survivin. One class ofagents will reduce or block the association by binding to thep34^(cdc2)-cyclin B1 kinase complex while another class of agents willreduce or block the association by binding to survivin.

As used herein, an agent is said to reduce or block phosphorylatedsurvivin/caspase-9 complex or unphosphorylated survivin/procaspase-9complex association when the presence of the agent decreases the extentto which or prevents the caspase from becoming associated with survivin.One class of agents will reduce or block the association by binding tocaspase-9 or procaspase-9 while another class of agents will reduce orblock the association by binding to survivin.

Agents that are assayed in the above methods can be randomly selected orrationally selected or designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences involved in the association of survivin with thep34^(cdc2)-cyclin B1 kinase complex or survivin with caspase-9. Anexample of randomly selected agents is the use a chemical library or apeptide combinatorial library, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. As described above, there are at least twosites of action for agents that block survivin/p34^(cdc2)-cyclin B1kinase complex interaction and survivin/caspase-9 interaction: thebinding partner contact site on survivin and the survivin contact siteon the kinase complex or on caspase-9. Agents can be rationally selectedor rationally designed by utilizing the peptide sequences that make upthe contact sites of survivin/p34^(cdc2)-cyclin B1 kinase complex and ofsurvivin/caspase-9 complex. For example, a rationally selected peptideagent can be a peptide whose amino acid sequence is identical to thesurvivin contact site on the kinase complex or on caspase-9. Such anagent will reduce or block the association of survivin with the kinasecomplex by binding to the p34^(cdc2)-cyclin. B1 kinase complex or theassociation of survivin by binding to caspase-9.

The agents of the present invention can be, as examples, peptides, smallmolecules, vitamin derivatives, as well as carbohydrates. A skilledartisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solidphase for solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

Another class of agents of the present invention are antibodiesimmunoreactive with critical positions of survivin, thep34^(cdc2)-cyclin B1 kinase complex, or caspase-9. Antibodies may beobtained by immunization of suitable mammalian subjects with peptides,containing as antigenic regions, those portions of the survivin orbinding partner, intended to be targeted by the antibodies. Criticalregions include the contact sites involved in the association ofsurvivin with the kinase complex, particularly, the region spanningamino acid residue 34 of survivin.

As used herein, survivin protein (or survivin) refers to a protein thathas the amino acid sequence of human survivin depicted in Ambrosini etal. (1997). The term “survivin protein” also includes naturallyoccurring allelic variants of survivin and naturally occurring proteinsthat have a slightly different amino acid sequence than thatspecifically recited above. Allelic variants, though possessing aslightly different amino acid sequence than those recited above, willstill have the requisite ability to inhibit cellular apoptosis.

As used herein, the survivin family of proteins refers to survivinproteins that have been isolated from organisms in addition to humans.The methods used to identify and isolate other members of the survivinfamily of proteins are readily available and described in applicationSer. No. 08/975,080.

The survivin proteins used in the assays or other embodiments of thepresent invention are preferably in isolated from. As used herein, aprotein is said to be isolated when physical, mechanical or chemicalmethods are employed to remove the survivin protein from cellularconstituents that are normally associated with the survivin protein. Askilled artisan can readily employ standard purification methods toobtain an isolated survivin protein.

The survivin proteins used in the present invention further includeconservative variants of the survivin proteins herein described. Aconservative variant refers to alterations in the amino acid sequencethat do not adversely affect the ability of the survivin protein to bindto a survivin binding partner, such as p34^(cdc2)-cyclin B1 kinasecomplex and caspase-9, and/or to inhibit cellular apoptosis. Asubstitution, insertion or deletion is said to adversely affect thesurvivin protein when the altered sequence prevents the survivin proteinfrom associating with a survivin binding partner and/or prevents thesurvivin protein or survivin protein from inhibiting cellular apoptosis.For example, the overall charge, structure or hydrophobic/hydrophilicproperties of survivin can be altered without adversely affecting theactivity of survivin. Accordingly, the amino acid sequence of survivincan be altered, for example to render the peptide more hydrophobic orhydrophilic, without adversely affecting the activity of survivin.

The allelic variants, the conservative substitution variants and themembers of the survivin family of proteins retain the ability to inhibitcellular apoptosis. Such proteins will ordinarily have an amino acidsequence having at least about 75% amino acid sequence identity with thehuman survivin sequence, more preferably at least about 80%, even morepreferably at least about 90%, and most preferably at least about 95%.Identity or homology with respect to such sequences is defined herein asthe percentage of amino acid residues in the candidate sequence that areidentical with the known peptides, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent homology,and including any conservative substitutions as being homologous.N-terminal, C-terminal or internal extensions, deletions, or insertionsinto the peptide sequence shall not be construed as affecting homology.

Homology or identity as used above is determined by BLAST (Basic LocalAlignment Search Tool) analysis using the algorithm employed by theprograms blastp, blastn, blastx, tblastn and tblastx (Karlin, et al.Proc. Natl. Acad. Sci. USA 87: 2264-2268 (1990) and Altschul, S. F. J.Mol. Evol. 36: 290-300 (1993), fully incorporated by reference) whichare tailored for sequence similarity searching. The approach used by theBLAST program is to first consider similar segments between a querysequence and a database sequence, then to evaluate the statisticalsignificance of all matches that are identified and finally to summarizeonly those matches which satisfy a preselected threshold ofsignificance. For a discussion of basic issues in similarity searchingof sequence databases, see Altschul et al. (Nature Genetics 6: 119-129(1994)) which is fully incorporated by reference. The search parametersfor histogram, descriptions, alignments, expect (i.e., the statisticalsignificance threshold for reporting matches against databasesequences), cutoff, matrix and filter are at the default settings. Thedefault scoring matrix used by blastp, blastx, tblastn, and tblastx isthe BLOSUM62 matrix (Henikoff, et al. Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992), fully incorporated by reference). For blastn, thescoring matrix is set by the ratios of M (i.e., the reward score for apair of matching residues) to N (i.e., the penalty score for mismatchingresidues), wherein the default values for M and N are 5 and −4,respectively.

The present invention also includes the use of survivin mimetics.Survivin mimetics are compounds that mimic the activity of survivinpeptides. They are structurally similar to survivin peptides but have achemically modified peptide backbone. Survivin mimetics may havesignificant advantages over polypeptide embodiments, including, forexample: more economical production; greater chemical stability;enhanced pharmacological properties (half-life, absorption, potency,efficacy, etc.); altered specificity (e.g., a broad-spectrum ofbiological activities); reduced antigenicity; and others.

Thus, the survivin proteins of the present invention include moleculeshaving the amino acid sequences disclosed in Ambrosini et al. (1997);fragments thereof having a consecutive sequence of at least about 3, 5,10, or 15 or more amino acid residues of the survivin protein; aminoacid sequence variants of such sequence wherein an amino acid residuehas been inserted N- or C-terminal to, or within, the survivin sequence;amino acid sequence variants of the survivin sequence, or theirfragments as defined above, that have been substituted by anotherresidue. Contemplated variants further include those containingpredetermined mutations by, e.g., homologous recombination,site-directed or PCR mutagenesis, the corresponding survivin proteins ofother animal species, including but not limited to rabbit, rat, murine,porcine, bovine, ovine, equine and non-human primate species, thealleles or other naturally occurring variants of the survivin family ofproteins; and derivatives wherein the survivin protein has beencovalently modified by substitution, chemical, enzymatic, or otherappropriate means with a moiety other than a naturally occurring aminoacid (for example a detectable moiety such as an enzyme orradioisotope). The recombinant survivin protein also can be used tosolve the molecular structure of survivin by 2D-NMR, circular dichroismand X-ray crystallography, thus integrating the site-directedmutagenesis approach and the rational design of specific small moleculeinhibitors.

As used herein, the term “caspase-9” includes any caspase-9 protein thatinteracts with phosphorylated survivin. The term includes the activatedform of the caspase-9, naturally occurring allelic variants ofcaspase-9, naturally occurring caspase-9 proteins isolated fromdifferent sources, variants of caspase-9 that interact with survivin,and fragments of caspase-9 that interact with survivin. As used herein,procaspase-9 refers to the proform of caspase-9 (see Zou, H. et al.,1999).

As used herein, the term “p34^(cdc2)-cyclin B1 kinase complex” refers toa complex comprising p34^(cdc2) and cyclin B1 kinase. The term“p34^(cdc2)” includes any p34^(cdc2) protein that forms a complex withcyclin B1 and the resulting complex phosphorylates and binds survivin.The term includes naturally occurring allelic variants of p34^(cdc2),naturally occurring p34^(cdc2) proteins isolated from different sources,variants of p34^(cdc2) that interact with survivin, and fragments ofp34^(cdc2) that interact with survivin. The term “cyclin B1” includesany cyclin B1 protein that forms a complex with p34^(cdc2) and theresulting complex phosphorylates and binds survivin. The term includesnaturally occurring allelic variants of cyclin B1, naturally occurringcyclin B1 proteins isolated from different sources, variants of cyclinB1 that interact with survivin, and fragments of cyclin B1 that interactwith survivin.

Assays of the invention may be modified or prepared in any availableformat, including high-throughput assays that monitor the binding ofsurvivin and p34^(cdc2)-cyclin B1 kinase complex or the binding ofsurvivin to caspase-9. In many drug screening programs which testlibraries of compounds, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity and/or bioavailability of the test compoundcan be generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an inhibition of, for instance, binding between tomolecules.

In one embodiment of a high-throughput screening assay survivin,p34^(cdc2)-cyclin B1 kinase complex, and caspase-9 may be added to thewells of a microtiter plate in the presence and absence of the agents tobe tested. Caspase-9 substrates that are commercially available could beincluded in the wells and the cleavage or release of the substrate canbe assayed using continuous-reading instruments as described in Quail etal. (1995) J. Biol. Chem. 270:10377-10379 or Stennicke et al. (1997) J.Biol. Chem. 272: 25719-25723.

In another embodiment of a high-throughput screening assay, the assaycan be formulated to detect the ability of a test compound to inhibitbinding, competitive or non-competitive, of survivin to thep34^(cdc2)-cyclin B1 kinase complex or of survivin to caspase-9. Theinhibition of complex formation may be detected by a variety oftechniques. For instance, modulation of the formation of complexes canbe quantitated using, for example, detectably labeled survivin such asradiolabeled (e.g. ³²P, ³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g.FITC), or enzymatically labeled survivin, by immunoassay, or bychromatographic detection.

To illustrate, there are a variety of binding assays known in the artfor detecting H2-receptor antagonists based on their ability to inhibitbinding of known H2 receptor ligands (including other antagonists). Inone embodiment, the in vitro assay described by Norris et al. ((1985)Agents Actions 16:170) can be used to score for substitutedN-heteroaromatics which bind to the H2-receptor (and which may befurther characterized in subsequent biological assays as agonists orantagonists of that receptor). In particular, the Norris et al. assayutilizes a competitive binding assay which detects inhibition of³H-tiotidine binding to guinea-pig cerebral cortex H2 receptors.

In certain assays, the receptor, subunits thereof, or even the othertarget protein to which binding is to be assessed, can be provided in apure or semi-pure form. Typically, for those instances, it will bedesirable to immobilize one of the proteins to facilitate separation ofprotein-protein complexes from uncomplexed forms, as well as toaccommodate automation of the assay. Binding of one protein to a secondprotein, for instance binding of survivin to p34^(cdc2)-cyclin B1 kinasecomplex or binding of survivin to caspase-9, in the presence and absenceof a candidate agent, can be accomplished in any vessel suitable forcontaining the reactants. Examples include microtitre plates, testtubes, and micro-centrifuge tubes. In one embodiment, a fusion proteincan be provided which adds a domain that allows the protein to be boundto a matrix. For example, glutathione-S-transferase (GST) fusionproteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the remaining labeled protein (ligand) andthe test compound. The mixture is then incubated under conditionsconducive to complex formation. Following incubation, the beads arewashed to remove any unbound ligand, and the matrix immobilized labeldetermined directly, or in the supernatant after the protein/ligandcomplexes are subsequently dissociated. When amenable, the complexes canbe dissociated from the matrix, separated by SDS-PAGE, and the level ofligand found in the bead fraction quantitated from the gel usingstandard electrophoretic techniques.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either protein canbe immobilized utilizing conjugation of biotin and streptavidin.Biotinylated molecules can be prepared from biotin-NHS(N-hydroxy-succinimide) using techniques well known in the art (e.g.,biotinylation kit, Pierce Chemicals, Rockford, Ill.), and immobilized inthe wells of streptavidin-coated 96 well plates (Pierce Chemical).Alternatively, antibodies reactive with the protein but which do notinterfere with ligand binding can be derivatized to the wells of theplate, and the first protein, such as polymerized tubulin, trapped inthe wells by antibody conjugation. As above, preparations of a ligandand a test compound are incubated in the protein-presenting wells of theplate, and the amount of protein/ligand complex trapped in the well canbe quantitated. Exemplary methods for detecting such complexes, inaddition to those described above, include immunodetection of complexesusing antibodies reactive with the ligand, or which are reactive withthe protein and compete for binding with the ligand.

Assays of the invention may also include any available in vivo basedscreening system to detect the interactions between two proteins. Forinstance, commonly available genetic systems are capable of rapidlydetecting which proteins interact with a known protein, determiningwhich domains of the proteins interact and identifying agents whichmodulate the interaction between two proteins. One such system is theyeast two-hybrid system wherein two proteins are expressed in yeast: oneprotein of interest fused to a DNA-binding domain and the other proteinof interest fused to a transcriptional activation domain (Fields et al.(1989) Nature 340:245; Gyuris et al. (1993) Cell 75:791; Harper et al.(1993) Cell 75:805; Serrano et al. (1993) Nature 366:704; and Hannon etal., (1993) Genes & Dev. 7:2378).

The amount of phosphorylated survivin can be quantitated by methodsroutinely practiced by the skilled artisan. One well-known methodemploys radiolabeling, electrophoresis, and scintillation counting.After electrophoresis of the phosphorylated samples on SDS-PAGE, the gelis autoradiographed and gel bands containing phosphorylated survivin areexcised and placed in a scintillation counter for determining the amountof phosphorylation (U.S. Pat. No. 6,028,171). Alternatively, thephosphorylated samples are precipitated with TCA and counted in ascintillation counter (U.S. Pat. No. 6,028,171). A second well-knownmethod uses an antibody that recognizes phosphorylated compounds.Example 1 discloses an example of an antibody that binds specifically tosurvivin phosphorylated at Thr34. The antibody does not recognizeunphosphorylated survivin. An antibody can be used to directlyimmunoprecipitate phosphorylated survivin from a sample. Afterimmunoprecipitation, the sample can be counted in a scintillationcounter. Also, immunoprecipitation with survivin antibody followed byelectrophoresis of immunoprecipitated sample, gel transfer ontonitrocellulose, and immunoblot with an antibody is another method ofusing anti-phosphotyrosine antibody to quantitate the amount ofphosphorylated compound (U.S. Pat. No. 5,635,388). Moreover, the amountof phosphorylated survivin can be determined by quantitativedensitometry. Recently, Angeles et al. developed a new method ofquantitating phosphorylation using an anti-phosphotyrosine antibody witha colorimetric readout or a lanthanide (europium)-labeledanti-phosphotyrosine antibody with a fluorometric detection (Angeles etal., Anal Biochem (2000), 278(2):93). Further, quantitation ofphosphorylated survivin can be performed using microtiter plates.

Kinase assays are useful as tools for in vitro detection of agents thatmodulate the binding of survivin and p34^(cdc2)-cyclin B1 kinase orsurvivin and caspase-9. The amount of phosphorylated survivin in thepresence and absence of the agent would enable the skilled artisan todetermine whether the agent inhibits binding of survivin andp34^(cdc2)-cyclin B1 kinase or survivin and caspase-9.

The above assays may be modified to screen for agents thatdephosphorylate survivin at Thr34. These agents would be expected to actas potential tumor suppressors, removing survivin phosphorylation andpreventing the formation of the anti-apoptotic complex with caspase. Theassay can be performed by modifying a standard in vitro kinase assay todetect or quantitate the amount of dephosphorylated survivin in thepresence of the agent and compared to that in the absence of the agent.Potential libraries could be screened for proteins that dephosphorylatesurvivin.

B. Apoptosis Assays

As a second step in the identification of agents which modulatephosphorylation of survivin, the interaction between survivin andp34^(cdc2)-cyclin B1 kinase complex, or the interaction between survivinand caspase-9, agents identified by the primary screen may then beevaluated in an apoptosis assay to determine the apoptotic activity ofthe agent. Specific examples of apoptosis assays are widely available inthe art as exemplified in the following references. Assays for apoptosisin lymphocytes are disclosed by: Li et al., (1995) Science 268:429-431;Gibellini et al. (1995) Br. J. Haematol. 89:24-33; Martin et al. (1994)J. Immunol. 152:330-42; Terai et al., (1991) J. Clin Invest. 87:1710-5;Dhein et al. (1995) Nature 373:438-441; Katsikis et al. (1995) J. Exp.Med. 1815:2029-2036; Westendorp et al. (1995) Nature 375:497; andDeRossi et al. (1994) Virology 198:234-44.

Assays for apoptosis in fibroblasts are disclosed by: Vossbeck et al.(1995) Int. J. Cancer 61:92-97; Goruppi et al. (1994) Oncogene9:1537-44; Fernandez et al. (1994) Oncogene 9:2009-17; Harrington et al.(1994) EMBO J., 13:3286-3295; and Itoh et al., (1993) J. Biol. Chem.268:10932-7.

Assays for apoptosis in neuronal cells are disclosed by: Melino et al.(1994) Mol. Cell. Biol. 14:6584-6596; Rosenblaum et al. (1994) Ann.Neural. 36:864-870; Sato et al. (1994) J. Neurobiol. 25:1227-1234;Ferrari et al. (1995) J. Neurosci. 1516:2857-2866; Talley et al. (1995)Mol. Cell Biol. 1585:2359-2366; Talley et al. (1995) Mol. Cell. Biol.15:2359-2366; and Walkinshaw et al. (1995) J. Clin. Invest.95:2458-2464.

Assays for apoptosis in insect cells are disclosed by: Clem et al.(1991) Science 254:1388-90; Crook et al. (1993) J. Virol: 67:2168-74;Rabizadeh et al. (1993) J. Neurochem. 61:2318-21; Birnbaum et al. (1994)J. Virol. 68:2521-8, 1994; and Clem et al. (1994) Mol. Cell. Biol.14:5212-5222.

C. Uses for Agents that Block the Association of Survivin withp34^(cdc2)-Cyclin B1 Kinase Complex

As provided in the Background section, survivin inhibits cellularapoptosis. Agents that reduce or block the phosphorylation of survivin,the interaction of survivin with p34^(cdc2)-cyclin B1 kinase complex, orthe interaction of survivin to caspase-9 can be used to modulatebiological and pathologic processes associated with survivin functionand activity.

In detail, a biological or pathological process mediated by survivin canbe modulated by administering to a subject an agent that blocks theinteraction of survivin with p34^(cdc2)-cyclin B1 kinase complex, thephosphorylation of survivin, or the interaction of survivin andcaspase-9.

As used herein, a subject can be any mammal, so long as the mammal is inneed of modulation of a pathological or biological process mediated bysurvivin. The term “mammal” is meant an individual belonging to theclass Mammalia. The invention is particularly useful in the treatment ofhuman subjects.

As used herein, a biological or pathological process mediated byphosphorylation survivin, survivin/p34^(cdc2)-cyclin B1 kinase complex,or survivin/caspase-9 interaction of a cell refers to the wide varietyof cellular events mediated by survivin. Pathological processes refer toa category of biological processes which produce a deleterious effect.For example, a pathological process mediated by survivin is theinhibition of cellular apoptosis in tumor cells. This pathologicalprocess can be modulated using agents that reduce or block survivinphosphorylation, survivin/p34^(cdc2)-cyclin B1 kinase complex binding,or survivin/caspase-9 binding.

As used herein, an agent is said to modulate a pathological process whenthe agent reduces the degree or severity of the process. For example, anagent is said to modulate tumor cell proliferation when the agentdecreases the rate or extent of cell division.

D. Methods of Inhibiting the Growth of Tumors Using Survivin Antagonists

As used herein, the term “survivin antagonist” encompasses any compoundthat antagonizes the expression of survivin or the activity of survivin.Examples of survivin antagonists include survivin mutants, survivinantibodies, survivin antisense nucleic acids, and any compound that willantagonize or inhibit the activity or the expression of survivin.Specific examples of survivin antagonists include but are not limited todominant-negative survivin mutant Thr³⁴→Ala and survivin antisensenucleic acid having a specific sequence.

An antisense survivin molecule is complementary to and capable ofhybridizing with the RNA encoded by a survivin gene (the “sense gene”).An antisense survivin molecule is used to inhibit the expression of thesurvivin gene thereby inhibiting tumor growth and preventing andtreating diseases associated with tumor growth.

Antisense nucleic acids are preferably constructed by inverting thecoding region of the sense gene relative to its normal presentation fortranscription to allow for transcription of its complement, hence thecomplementariness of the respective RNAs encoded by these DNA's. Inorder to block the production of mRNA produced by the sense gene, theantisense DNA should preferably be expressed at approximately the sametime as the sense gene if the antisense nucleic acid is to be expressedin the cell. The timing must be approximate in the sense that theantisense RNA must be present within the cell to block the function ofthe RNA encoded by the sense gene. To accomplish this result, the codingregion of the antisense DNA is often placed under the control of thesame promoter as found in the sense gene thereby causing both to betranscribed at the same time.

For reviews of the design considerations and use of antisenseoligonucleotides, see Uhlmann et al. (1990) and Milligan et al. (1993),the disclosures of which are hereby incorporated by reference.

While in principle, antisense nucleic acids having a sequencecomplementary to any region of the survivin gene may be useful in thetumor growth inhibition methods of the present invention, nucleic acidmolecules complementary to a portion of the survivin mRNA transcriptincluding the translation initiation codon are particularly preferred.Also preferred are nucleic acid molecules complementary to a portion ofthe survivin mRNA transcript lying within about 40 nucleotides upstream(the 5′ direction) or about 40 nucleotides downstream (the 3′ direction)from the translation initiation codon.

In another embodiment, antisense oligonucleotides which hybridize oranneal to at least a portion of the survivin mRNA in a cell may be usedin the methods of the invention. Such oligonucleotides are typicallyshort in length and fairly easily diffusible into a cell. Such antisenseoligonucleotides include, but are not limited to, polydeoxynucleotidescontaining 2′-deoxy-D-ribose, polyribonucleotides containing D-ribose,any other type of polynucleotide which is an N-glycoside of a purine orpyrimidine base, or other polymers containing nonnucleotide backbones(e.g., protein nucleic acids and synthetic sequence specific nucleicacid polymers commercially available) or nonstandard linkages, providingthat the polymers contain nucleotides in a configuration which allowsfor base pairing and base stacking such as is found in DNA and RNA. Theymay include double- and single-stranded DNA, as well as double- andsingle-stranded RNA and DNA:RNA hybrids, and also include, as well asunmodified forms of the polynucleotide or oligonucleotide, known typesof modifications, for example, labels which are known to those skilledin the art, “caps”, methylation, substitution of one or more of thenaturally occurring nucleotides with analogue, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphorotriesters, phosphoramidates, carbamates,etc.) and with charged linkages or sulfur-containing linkages (e.g.,phosphorothioates, phosphorodithioates, etc.), those containing pendantmoieties, such as, for example, proteins (including nucleases, nucleaseinhibitors, toxins, antibodies, signal peptides, poly-L-lysine, etc.)and saccharides (e.g., monosaccharides, etc.), those with intercalators(e.g., acridine, psoralen, etc.), those containing chelators (e.g.,metals, radioactive metals, boron, oxidative metals, etc.), thosecontaining alkylating agents, those with modified linkages (e.g., alphaanomeric nucleic acids, etc.).

The terms “nucleoside”, “nucleotide” and “nucleic acid” as usedconcerning survivin antisense nucleic acid molecules, include thosemoieties which contain not only the known purine and pyrimidine bases,but also other heterocyclic bases which have been modified. Suchmodifications include methylated purines and pyrimidines, acylatedpurines and pyrimidines, or other heterocycles. Modified nucleosides ornucleotides will also include modifications on the sugar moiety, e.g.,wherein one or more of the hydroxyl groups are replaced with halogen,aliphatic groups, or are functionalized as ethers, amines, or the like.

The present invention provides a method of inhibiting tumor growth byadministering a survivin antagonist to the site of tumor growth. Asshown in the examples, subclones of YUSAC-2 human melanoma cells stablytransfected with survivin antagonist, survivin antisense or adominant-negative survivin mutant (Thr³⁴→Ala) under the control of atetracycline (tet)-regulated promoter were generated. Cells expressingthese survivin antagonists underwent spontaneous apoptosis in vitro anddid not form tumors upon subcutaneous injection into CB.17 mice.Expression of survivin Thr³⁴→Ala mutant in established tumors slowedtheir growth and caused apoptosis and aberrant mitotic progression inmelanoma cells. Manipulation of the apoptotic pathway by targetingsurvivin may be beneficial in cancer therapy. The survivin antagonistsof the present invention can be used to treat patients diagnosed withcancer.

E. Administration of Agents that Modulate Survivin/p34^(cdc2)-cyclin B1Kinase Complex Interactions and Survivin/Caspase-9 Interactions

The agents of the present invention, such as agents that block survivinphosphorylation, survivin/p34^(cdc2)-cyclin B1 kinase complexassociation, or survivin/caspase-9 interaction can be administered viaparenteral, subcutaneous, intravenous, intramuscular, intraperitoneal,transdermal, or buccal routes. Alternatively, or concurrently,administration may be by the oral route. The dosage administered will bedependent upon the age, health, and weight of the recipient, kind ofconcurrent treatment, if any, frequency of treatment, and the nature ofthe effect desired. For example, as a means of blocking survivininhibition of apoptosis in tumor cells or to inhibit survivin inducedangiogenesis, an agent that blocks survivin phosphorylation,survivin/p34^(cdc2)-cyclin B1 kinase complex association is administeredsystemically or locally to the individual being treated. As describedbelow, there are many methods that can readily be adapted to administersuch agents.

The present invention further provides compositions containing one ormore agents that block phosphorylation of survivin,survivin/p34^(cdc2)-cyclin B1 kinase complex association, orsurvivin/caspase-9 association. While individual needs vary, adetermination of optimal ranges of effective amounts of each componentin the composition is within the skill of the art. Typical dosagescomprise 0.1 to 100 μg/kg body wt. The preferred dosages comprise 0.1 to10 μg/kg body wt. The most preferred dosages comprise 0.1 to 1 μg/kgbody wt.

In addition to the pharmacologically active agent, the compositions ofthe present invention may contain suitable pharmaceutically acceptablecarriers comprising excipients and auxiliaries which facilitateprocessing of the active compounds into preparations which can be usedpharmaceutically for delivery to the site of action.

Suitable formulations for parenteral administration include aqueoussolutions of the active compounds in water-soluble form, for example,water-soluble salts. In addition, suspensions of the active compounds asappropriate oily injection suspensions may be administered. Suitablelipophilic solvents or vehicles include fatty oils, for example, sesameoil, or synthetic fatty acid esters, for example, ethyl oleate ortriglycerides. Aqueous injection suspensions may contain substanceswhich increase the viscosity of the suspension include, for example,sodium carboxymethyl cellulose, sorbitol, and/or dextran. Optionally,the suspension may also contain stabilizers. Liposomes can also be usedto encapsulate the agent for delivery into the cell.

The pharmaceutical formulation for systemic administration according tothe invention may be formulated for enteral, parenteral or topicaladministration. Indeed, all three types of formulations may be usedsimultaneously to achieve systemic administration of the activeingredient.

Suitable formulations for oral administration include hard or softgelatin capsules, pills, tablets, including coated tablets, elixirs,suspensions, syrups or inhalations and controlled release forms thereof.

In practicing the methods of this invention, the compounds of thisinvention may be used alone or in combination, or in combination withother therapeutic or diagnostic agents. In certain preferredembodiments, the compounds of this invention may be coadministered alongwith other compounds typically prescribed for these conditions accordingto generally accepted medical practice, such as chemotherapeutic agents.

F. Methods of Delivering a Survivin Antagonists

Survivin antagonists that are not nucleic acid molecules can bedelivered to target sites as discussed under Section E, above. Survivinantagonists that are nucleic acid molecules can be delivered to targetsites as discussed below.

Gene therapy is a method for delivering functionally active therapeuticor other forms of genes into targeted cells. Initial efforts of genetransfer into somatic tissues have relied on indirect means called exvivo gene therapy, wherein target cells are removed from the body,transfected or infected with vectors carrying recombinant genes, andre-implanted into the body. Techniques currently used to transfer DNA invitro into cells include calcium phosphate-DNA precipitation,DEAE-Dextran transfection, electroporation, liposome mediated DNAtransfer or transduction with recombinant viral vectors. Thesetransfection protocols have been used to transfer DNA into differentcell types including epithelial cells (U.S. Pat. No. 4,868,116; Morganet al., 1987), endothelial cells (WO89/05345), hepatocytes (Ledley etal., 1987; Wilson et al., 1990) fibroblasts (Rosenberg et al., 1988;U.S. Pat. No. 4,963,489), lymphocytes (U.S. Pat. No. 5,399,346; Blaeseet al., 1995) and hematopoietic stem cells (Lim et al., 1989; U.S. Pat.No. 5,399,346).

Direct in vivo gene transfer has been carried out with formulations ofDNA trapped in liposomes (Ledley et al., 1987), or in proteoliposomesthat contain viral envelope receptor proteins (Nicolau et al., 1983),and with DNA coupled to a polylysine-glycoprotein carrier complex. Inaddition, “gene guns” have been used for gene delivery into cells(Australian Patent No. 9068389). Lastly, naked DNA, or DNA associatedwith liposomes, can be formulated in liquid carrier solutions forinjection into interstitial spaces for transfer of DNA into cells(WO90/11092).

Viral vectors are often the most efficient gene therapy system, andrecombinant replication-defective viral vectors have been used totransduce (i.e., infect) cells both ex vivo and in vivo. Such vectorsinclude retroviral, adenovirus and adeno-associated and herpes viralvectors. Accordingly, in one embodiment the survivin transgene orsurvivin antisense molecule can be subcloned into an appropriate vectorand transferred into a cell or tissue by gene transfer techniquesdiscussed above.

In another embodiment, the survivin antisense molecule can be providedto the cell or tissue using a transfection facilitating composition,such as cationic liposomes containing desired polynucleotide.

G. Combination Therapy

Agents and survivin antagonists of the present invention, can beprovided alone, or in combination with other agents that modulate aparticular biological or pathological process. For example, an agent ofthe present invention that inhibits survivin/p34^(cdc2)-cyclin B1 kinasecomplex association, survivin phosphorylation, or survivin/caspase-9interaction can be administered in combination with anti-cancer agentsin methods to control cancer cell growth. Likewise, a survivinantagonist can be administered in combination with anti-cancer agents toinhibit growth of tumor. As used herein, two agents are said to beadministered in combination when the two agents are administeredsimultaneously or are administered independently in a fashion such thatthe agents will act at the same time.

Inhibition of survivin activity can be used in combination withconventional chemotherapies or anti-angiogenesis agents. The timing forusing such agents in combination with agents that inhibit survivinactivity depends upon the chemotherapeutic agent used and the tumor celltype treated. Examples of chemotherapeutic agents that can be used incombination with agents the effect survivin activity, include, but arenot limited to alkylating agents, such as cyclophosphamide (CTX;cytoxan), chlorambucil (CHL; leukeran), cisplatin (CisP; platinol)busulfan (myleran), melphalan, carmustine (BCNU), streptozotocin,triethylenemelamine (TEM), mitomycin C, and the like alkylating agents;anti-metabolites, such as methotrexate (MTX), etoposide (VP16; vepesid)6-mercaptopurine (6MP), 6-thiocguanine (6TG), cytarabine (Ara-C),5-fluorouracil (5FU), dacarbazine (DTIC), and the like anti-metabolites;antibiotics, such as actinomycin D, doxorubicin (DXR; adriamycin),daunorubicin (daunomycin), bleomycin, mithramycin and the likeantibiotics; alkaloids, such as vinca alkaloids such as vincristine(VCR), vinblastine, and the like; and other antitumor agents, such astaxol and taxol derivatives, the cytostatic agents glucocorticoids suchas dexamethasone (DEX; decadron) and corticosteroids such as prednisone,nucleoside enzyme inhibitors such as hydroxyurea, amino acid depletingenzymes such as asparaginase, and the like diverse antitumor agents.

The use of the cytotoxic agents described above in chemotherapeuticregimens is generally well characterized in the cancer therapy arts, andtheir use herein falls under the same considerations for monitoringtolerance and effectiveness and for controlling administration routesand dosages, with some adjustments. For example, the actual dosages ofthe cytotoxic agents may vary depending upon the patient's cultured cellresponse determined by using the present histoculture methods.Generally, the dosage will be reduced compared to the amount used in theabsence of agents the effect Survivin activity/expression.

Typical dosages of an effective cytotoxic agent can be in the rangesrecommended by the manufacturer, and where indicated by in vitroresponses or responses in animal models, can be reduced by up to aboutone order of magnitude concentration or amount. Thus, the actual dosagewill depend upon the judgment of the physician, the condition of thepatient, and the effectiveness of the therapeutic method based on the invitro responsiveness of the primary cultured malignant cells orhistocultured tissue sample, or the responses observed in theappropriate animal models.

Without further description, it is believed that one of ordinary skillin the art can, using the preceding description and the followingillustrative examples, make and utilize the compounds of the presentinvention and practice the claimed methods. The following workingexamples therefore, specifically point out preferred embodiments of thepresent invention, and are not to be construed as limiting in any waythe remainder of the disclosure. All articles, publications, patents anddocuments referred to throughout this application are herebyincorporated by reference in their entirety.

EXAMPLES Materials and Methods

Cell lines. Cervical carcinoma HeLa cells (American Type CultureCollection) were maintained in Dulbecco's modified minimal Eagle'smedium (DMEM; GIBCO BRL, Grand Island, N.Y.) medium, containing 10%fetal calf serum (FCS, Gemini Bio-Products, Calabasas, Calif.), andantibiotics. The YUSAC-2 melanoma cell line expressing endogenoussurvivin was characterized previously (Grossman et al., 1999).Cell-cycle synchronization was carried out as described (Li et al.,1998), by culture in the presence of 400 μM mimosine (G1), 2 mMthymidine (S) or 0.4 μg/ml nocodazole (G2/M), for 16-h at 37 C. Cellcycle arrest under the various conditions was confirmed by DNA contentanalysis by propidium iodide staining and flow cytometry.

Antibodies. An affinity-purified rabbit antibody to wild typerecombinant survivin was described previously (Grossman et al., 1999).Anti-survivin mAb 8E2 (IgG1) was used in immunofluorescence, asdescribed (Li et al., 1998), Rabbit antibodies to caspase-9 (1:2000dilution) and caspase-8 (1 μg/ml) were purchased from Pharmingen, SanDiego, Calif. Rabbit antibodies to p34^(cdc2)(1-10 μg/ml) and Cdk2 (10μg/ml) were purchased from Zymed, San Francisco, Calif., and Santa Cruz,respectively. A rat antibody to HA (0.1-5 μg/ml) was from BoehringerMannheim, San Diego, Calif. A rabbit antibody was raised against thesurvivin peptide sequence L²⁸EGCACT*PERMAEAGFI⁴⁴ containingphosphorylated Thr³⁴(*), according to published protocols (Grossman etal., 1999). Aliquots of the immune serum were precleared by overnightincubation with Sepharose 2B under constant agitation at 4° C., appliedto a Sepharose column containing the parent non-phosphorylated survivinpeptide L²⁸EGCACTPERMAEAGFI⁴⁴, and unbound material was further affinitypurified on a phosphorylated L²⁸EGCACT*PERMAEAGFI⁴⁴ survivin peptidecolumn. Bound immunoglobulins were eluted in 0.1 M glycine, pH 2.5,neutralized, dialyzed overnight against PBS, pH 7.4, and used inimmunoblotting experiments at 10 μg/ml. Antibodies that recognizephosphorylated survivin can be used in the methods of the invention andmay also be used to monitor the efficacy of treatment with an antagonistof survivin phosphorylation or may be used to detect phosphorylatedsurvivin in cell samples, such as tumor samples ny immunohistochemistryor Western blot.

Oligonucleotides, plasmids and recombinant proteins. Site-directedmutagenesis of the wild type survivin cDNA (Ambrosini et al., 1997) wascarried out using the GeneEditor system (Promega, Madison, Wis.), withthe targeting oligonucleotide 5′GGCTGCGCCTGCgCCCCGGAGCGGATG3′ to insertthe Thr³⁴→Ala (T34A) substitution, according to the manufacturer'sspecifications. After antibiotic selection, the mutated plasmid DNA wasisolated, and confirmed by DNA sequencing. A survivin (T34A) cDNA wasalso separately inserted in the HindIII/EcoRI sites of pcDNA3(Invitrogen, San Diego, Calif.) and pEGFPc1 (Clontech, San Francisco,Calif.) vectors. HA-tagged constructs encoding wild type survivin andsurvivin (T34A) were obtained by PCR using a 5′-oligonucleotide5′CCCAAGCTTATGTATCCGTATGATGTTCCTGATTATGCTGGTGCCCCGACGT TGCCC3′containing the HA tag and a 3′-oligonucleotide5′CGGGATCCGGAAGTGGTGCAGCCACTCTG3′ (wild type survivin) or5′ACGAATTCAATCCATGGCAGCCAG3′ (survivin (T34A)). PCR products weredigested with either HindIII/BamHI (522 bp, HA-survivin), orHindIII/EcoRI (473 bp, HA-survivin (T34)), directionally cloned inpcDNA3, and confirmed by DNA sequencing. A wild type survivin cDNA inpEGFPc1 was characterized previously (Li et al., 1999).

A caspase-9 cDNA encoding Met¹-Asp³³⁰ was amplified by PCR witholigonucleotides 5′ (5′CCCAAGCTTCCATGGACGAAGCGGATCGG3′, forward) and(5′CGGAATTCTTAGTCCAGCTGGTCGAAGGTC3′, reverse), digested withHindIII/EcoRI, directionally cloned in pEGFPc1, and confirmed by DNAsequencing. A caspase 9 dominant negative mutant (Cys²⁸⁷→Ala) wasgenerated by overlapping PCR by amplifying a 5′ product of the caspase-9cDNA of 892 nt with oligonucleotides 5′CCCAAGCTTCCATGGACGAAGCGGATCGG3′(forward) and 5′GTCTTTCTGCTCCCCACCggcGGCCTGGATGAAAAAGAGC3′ (reverse),and a 3′ product of 422 nt with oligonucleotides5′GCTCTTTTTCATCCAGGCCgccGGTGGGGAGCAGAAAGAC3′ (forward) and5′CGGAATTCTTATGATGTTTTAAAGAAAAGTTTTTTCC3′ (reverse). Equalconcentrations of each gel-purified caspase 9 fragment were amplified byPCR using oligonucleotides 5′CCCAAGCTTCCATGGACGAAGCGGATCGG3′ (forward)and 5′CGGAATTCTTATGATGTTTTAAAGAAAATTTTTTTCC3′ (reverse). The resultingproduct of 1272 nt PCR was digested with HindIII/EcoRI, directionallycloned in pcDNA3 or pEGFPc1 and confirmed by DNA sequencing, Allplasmids were purified by ion-exchange chromatography (Qiagen, Valencia,Calif.). Wild type survivin or survivin (T34A) in pGEX2T (Pharmacia,Piscataway, N.J.) were expressed as GST fusion proteins in BL21 E. Colias described previously (Li et al., 1998). The recombinant proteins werereleased from the GST frame by overnight cleavage with thrombin followedby neutralization in 1-M benzamidine and overnight dialysis in PBS, pH7.4. A caspase-8 (C360S) dominant negative mutant (Boldin et al., 1996)was obtained from Dr. V. Dixit (Genentech Inc. San Francisco).

Transfection experiments. For transient transfection, HeLa cells in C-6tissue culture plates (Costar, Cambridge, Mass.) at ˜60-70% confluency,were incubated in 1 ml of serum-free OptiMEM medium (Life Technologies,Gaithersburg, Md.) for 20 min, and transfected with 2.5 μg of thevarious plasmid cDNAs plus 9 μl Lipofectamine (Life Technologies). Aftera 5-h incubation at 37 C, the mixture was aspirated and substituted withcomplete growth medium for 48-72 h. To generate stable YUSAC-2transfectants, a survivin (T34A) cDNA was inserted into the HindIII-SpeIsites of the pTet-splice vector (Dr. D. Schatz, Yale University Schoolof Medicine), downstream of the regulatory sequences of theTet-resistance operon (TetO). YUSAC-2 cells were transfected with 0.8 μgpTet-T34A, 0.8 μg of the transactivation/selection plasmid ptTA-Neo and5 μl Lipofectamine in 1 ml of OptiMem. Forty-eight h after transfection,cells were re-plated at low density in 15×150 mm plates in growth mediumcontaining 1.5 mg/ml Geneticin (G418, Life Technologies), 2 mM sodiumhydroxide and 0.5 μg/ml Tet (Sigma, St. Louis, Mo.) (selection medium).After a 3-week culture, ninety-six individual clones were screened fordifferential growth in the presence or absence of Tet. Three clonesexhibited no growth in the absence of Tet, and one of them (F5.C4) wasre-cloned by limiting dilution.

Flow cytometry. HeLa cells transfected with the various GFP constructsin the presence or in the absence of the broad-spectrum caspaseinhibitor Z-VAD-fink (20 μM) were harvested after 48-h (non-adherent andadherent cells), washed in PBS, pH 7.0, and fixed in cold 70% ethanolfor 30 min on ice. After washes in PBS, cells (10×10₆/ml) were stainedwith 25 μg/ml propidium iodide (Sigma), 0.05% Triton X-100 and 100 μg/mlRNAse A (Boehringer Mannheim, Indianapolis, Ind.) for 45 min at 22 C.Gated GFP-expressing cells were analyzed for DNA content by flowcytometry on a FACSort (Becton Dickinson, San Jose, Calif.) using a CellQuest software. In other experiments, F5-C4 cells were synchronized atthe G1/S boundary by culture with 5-mM thymidine for 16 h at 37° C., inthe presence or in the absence of Tet. At the end of the incubation,cells were released from the thymidine block, and harvested at 3-hintervals for DNA content analysis by propidium iodide staining and flowcytometry, or, alternatively, for caspase-9 processing by Westernblotting (see below).

TUNEL staining. Internucleosomal DNA fragmentation of F5.C4 cells in thepresence or in the absence of Tet was carried out by end-labeling withterminal deoxynucleotidyl transferase (TdT) and peroxidase-conjugatedanti-digoxigenin antibody using the ApopTag kit (Intergen, Purchase,N.Y.), as described previously (Grossman et al., 1999).

Immunoprecipitation and Western blotting, Asynchronously growing orsynchronized HeLa cells were lysed in 200 μl of lysis buffer containing50 mM Tris, pH 7.5, 1% NP-40, 0.25% DOC, 150 mM NaCl, 1 mM PMSF, 10μg/ml aprotinin, 10 μg/ml leupeptin, 10 μg/ml pepstatin, 1 mM Na3Vo4, 20mM NaF, 0.2 mM EGTA, 1 mM EDTA, pH 8.0, for 30 min at 4° C. Insolublematerial was removed by centrifugation at 14,000 rpm for 15 min, andtwo-hundred μg of protein lysate was pre-absorbed with 25 μl of a 50:50Protein A Sepharose slurry (Pharmacia) for 3 h at 4° C. with constantagitation. Precleared lysates were separately incubated with antibodiesto p34^(cdc2), Cdk2, or HA for 16 h at 4° C. with constant agitation.The immune complexes were precipitated by addition of 50 μl of a 50:50Protein A slurry for 2 h at 4° C., washed three times in 350 mMNaCl/lysis buffer, plus three additional washes in 150 mM NaCl/lysisbuffer. Samples were separated by electrophoresis on a 12%SDS-polyacrylamide gel, transferred to Immobilon membranes (MilliporeCorp.), and separately incubated with antibodies to p34_(cdc2) (μg/ml),caspase-9, caspase-8, survivin, or HA (0.1 μg/ml) in TBS plus Tween-20for 16 h at 4° C. After washes, the transfer membranes were separatelyincubated with HRP-conjugated anti-mouse (Amersham), anti-rabbit(Amersham), or anti-rat (Boehringer) secondary antibodies (1:2000), withvisualization of immunoreactive bands by chemiluminescence (Amersham).For reprobing, membranes were stripped in 100 mM 2-β mercaptoethanol, 2%SDS, 62.5 mM Tris-HCl, pH 6.7, for 30 min at 50° C. In another series ofexperiments, HeLa cells at 60% confluency were transfected withHA-survivin or HA-survivin (T34A) and replenished with complete growthmedium for 8-h at 37 C. Cells were synchronized at the G1/S boundary bytreatment with 2 mM thymidine for 16 h at 37 C, released in completegrowth medium and harvested at increasing time intervals forimmunoprecipitation and DNA content analysis by flow cytometry, asdescribed above.

Survivin phosphorylation and in vitro kinase assays. Exponentiallygrowing HeLa cells were transiently transfected with HA-survivin andallowed to recover for 44 h in complete DMEM. Cells were supplementedwith phosphate-free DMEM (Life Technologies) for 1 h at 37° C. Cellswere labeled with 200 μCi/ml _(32 PI) (NEN Life Science Products Inc.)in the presence of 10% phosphate-free serum for an additional 5 h at 37°C. Labeled cells were washed twice in cold PBS, pH 7.4, solubilized inlysis buffer, and immunoprecipitated with an antibody to HA (see above),followed by autoradiography. For in vitro kinase assays,baculovirus-expressed human p34_(cdc2)-cyclin B1 or Cdk2-cyclin E kinasecomplexes were separately incubated with 1 μg histone H1, 6 μg of wildtype survivin or survivin (T34A) in a 25 μl reaction mixture containing20 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.5 mM DTT, 10 μM ATP plus 10 μCi of³²P-ATP (Amersham) for 30 min at 30° C. The reaction was terminated byaddition of 25 μl of 2× Laemmli sample buffer. Samples wereelectrophoresed on a 5-20% SDS polyacrylamide gel, and phosphorylatedbands were visualized by autoradiography. Equal protein loading in eachkinase reaction was confirmed by Coomassie blue staining of the gel.

Immunofluorescence and confocal microscopy. HeLa cells transfected withthe various GFP constructs were harvested after 48 h, and fixed in 4%paraformaldehyde containing 0.25% Triton X-100 for 10 min at 22 C. Cellnuclei were stained with 6.5 μg/ml 4,6-diamidino-2-phenylindole (DAPI,Sigma), 16% polyvinyl alcohol (Air Products and Chemicals, Allentown,Pa.), and 40% glycerol. GFP-expressing cells were independently scoredfor nuclear morphology of apoptosis (chromatin condensation, DNAfragmentation) using a Zeiss fluorescent microscope, as described (Li etal., 1999). For confocal microscopy analysis, HeLa cells on opticalgrade glass coverslips were fixed and labeled with mAb 8E2 to survivinor a rabbit antibody to p34_(cdc2), as described (Li et al., 1998).Binding of the primary antibodies was revealed by addition ofFITC-labeled goat anti-mouse IgG (survivin) and Texas Red(TR)-conjugated goat anti-rabbit IgG (p34_(cdc2)) (Molecular Probes,Junction City, Oreg.). In other experiments, HeLa cells were transfectedwith HA-survivin, HA-survivin (T34A), or survivin (L64A) and stainedwith antibodies to HA, mAb 8E2 (L64A), or caspase-9, followed byanti-mouse (FITC) or anti-rabbit (TR)-conjugated antibodies,respectively. For all experiments, non-binding mouse or rabbit IgG wereused as controls. Coverslips mounted in Mowiol 4-88 (Hoechst,Frankfurt/Main, Germany) were analyzed on a Zeiss Axiophot microscope orby confocal laser scanning microscopy (CLSM Bio-Rad 1024). Files wereassembled and printed with ADOBE Photoshop 5.0.

Example 1 Phosphorylation of Survivin

A. Phosphorylation of Survivin on Thr³⁴ by p34^(cdc2)-cyclin B1.

Inspection of the primary sequence of survivin revealed a potentialphosphorylation site for p34^(cdc2) at Thr³⁴(FIG. 1A). By Clustalalignment, the region T³⁴-P-E-R, which matched the p34^(cdc2) consensusphosphorylation sequence S/T-P-X-R (Holmes and Solomon, 1996), was foundonly in human and mouse survivin, and was absent in IAP proteins fromvarious species (FIG. 1A and Deveraux and Reed, 1999). In in vitrokinase assays, baculovirus-expressed p34^(cdc2)-cyclin B1 readilyphosphorylated recombinant wild type survivin, whereas substitution ofThr³⁴→Ala, i.e. survivin (T34A), abolished phosphorylation byp34^(cdc2)-cyclin B1 (FIG. 1B). In control experiments,baculovirus-expressed Cdk2-cyclin E did not phosphorylate wild typesurvivin or survivin (T34A), whereas p34′2-cyclin B1 or Cdk2-cyclin Ereadily phosphorylated histone H1 (FIG. 1B). A rabbit antibody (ATCC No.______) was raised against the survivin peptide sequenceL²⁸EGCACT*PERMAEAGFI⁴⁴ (SEQ ID NO: 1) containing phosphorylated Thr³⁴(T*), sequentially affinity purified onnon-phosphorylated/phosphorylated peptide-Sepharose columns and used inimmunoblotting. The antibody to phosphorylated Thr³⁴ recognized wildtype survivin after in vitro phosphorylation by p34^(cdc2)-cyclin B1,but not unphosphorylated survivin or survivin (T34A) after incubationwith p34^(cdc2)-cyclin B1 (FIG. 1C). In contrast, an antibody tosurvivin (Grossman et al., 1999) indistinguishably recognized wild typesurvivin or survivin (T34A), with or without p34^(cdc2)-cyclinB1-mediated phosphorylation (FIG. 1D). In other experiments, HA-taggedsurvivin was transfected in HeLa cells metabolically labeled with ³²Piorthophosphate. Immunoprecipitation with an antibody to HA demonstratedprominent phosphorylation of 16.5 kDa survivin, in vivo, whereas acontrol non-binding antibody did not immunoprecipitate radiolabeledbands from HeLa cells (FIG. 1D).

B. Physical Association Between Survivin and p34^(cdc2).

HeLa cells arrested at G1, S, or G2/M were detergent-solubilized andimmunoprecipitated with an antibody to p34^(cdc2). Western blotting ofp34^(cdc2) immunoprecipitates from G2/M-arrested HeLa cells revealed thepresence of associated 16.5 kDa survivin, which was enriched in mitoticcells as compared with asynchronously growing cultures (FIG. 2A). Incontrast, no survivin bands were immunoblotted in p34^(cdc2)immunoprecipitates from G1- or S-arrested cultures (FIG. 2A). Consistentwith the specificity of p34^(cdc2) phosphorylation of survivin (FIG. 1),Cdk2 immunoprecipitates from G1-, S-, or G2/M-synchronized HeLa cellsdid not contain 16.5 kDa survivin by Western blotting (FIG. 2A). Inreciprocal experiments, HA-survivin or HA-survivin (T34A)immunoprecipitated from HeLa cells contained a prominent p34^(cdc2) bandby Western blotting (FIG. 2B), thus demonstrating thatsurvivin-p34^(cdc2) interaction was independent of Thr³⁴. By dualimmunofluorescence labeling and confocal microscopy, endogenousp34^(cdc2) and survivin strongly co-localized on mitotic spindlemicrotubules at prophase and metaphase, and concentrated withinmidbodies at late telophase (FIG. 2C).

Example 2 Survivin Regulation of Apoptosis

A. Spontaneous Apoptosis Induced by Expression of Non-PhosphorylatableSurvivin (T34A).

Transfection of HeLa cells with survivin (T34A) fused to a GreenFluorescent Protein (GFP) caused spontaneous chromatin condensation andDNA fragmentation in ˜80% of GFP-expressing cells, in the absence ofexogenous apoptotic stimuli (FIG. 3A, B). Similar results were obtainedwith transfection of GFP-caspase-9 (Met¹)-Asp³³⁰, whereas expression ofGFP vector or wild type survivin did not affect nuclear morphology inHeLa cells (FIG. 3A, B). Comparable expression of the various constructsin HeLa cells was confirmed by flow cytometry by gating on theGFP-expressing population (not shown). In other experiments, expressionof GFP-survivin (T34A) resulted in the appearance of HeLa cells withhypodiploid (sub-G1) DNA content by propidium iodide staining and flowcytometry, in a reaction reversed by the broad-spectrum caspaseinhibitor, Z-VAD-fink (FIG. 3C). Immunofluorescence labeling of HeLacells expressing survivin (T34A) with an antibody to β-tubulin revealeda profound dysregulation of mitotic transitions, characterized byassembly of multipolar and grossly abnormal mitotic spindles (FIG. 3D).In contrast, HeLa cells transfected with wild type survivin formednormal bipolar spindles (FIG. 3D).

Apoptosis Induced by Survivin (T34A) Coincides with Mitosis.

YUSAC-2 melanoma cells expressing endogenous survivin (Grossman et al.,1999) were stably transfected to express survivin (T34A) under thecontrol of a tetracycline (Tet)-regulated promoter (Tet-off system)(Shockett et al., 1995). Withdrawal of Tet from three independenttransfected cell lines (Tet−) resulted in strong induction of ˜16.5 kDasurvivin (T34A) by Western blotting, but not in Tet-containing cultures(Tet+) (not shown). After induction of survivin (T34A), Tet-cell linesexhibited a nearly complete loss of mitotic (G2/M) cells, and appearanceof a prominent population with apoptotic (sub-G1) DNA content, bypropidium iodide staining and flow cytometry (FIG. 4A). In contrast,Tet+ lines had normal G2/M DNA content and did not exhibit increase inthe apoptotic (sub-G1) fraction (FIG. 4A). One transfected YUSAC-2 cellline (F5.C4) was re-cloned by limiting dilution and used in subsequentexperiments. Consistent with genuine apoptotic morphology (FIG. 2),F5.C4 cells strongly labeled for internucleosomal DNA fragmentation byTUNEL in the absence, but not in the presence of Tet (FIG. 4B).

F5.C4 cells were synchronized at the G1/S boundary with or without Tet,released, and monitored for cell cycle transitions at 3-h intervals bypropidium iodide staining and flow cytometry. In the presence of Tet(Tet+), F5.C4 cells approached the first mitosis 9 h after thymidinerelease, completed cell division by 15-18 h, and re-entered G1 after 21h, without significant changes in the apoptotic (sub-G1) fractionthroughout the cell cycle (FIG. 4C). In the absence of Tet, (Tet−) F5.C4cells exhibited similar kinetics of cell cycle progression (FIG. 4C).However, entry into mitosis of Tet-F5.C4 cells coincided precisely withthe appearance of the apoptotic population with sub-G1 DNA content,which increased steadily throughout mitosis and in the post-mitoticphase with considerable reduction of the subsequent G1 peak (FIG. 4C).

Example 3 Survivin and Capspase-9

Phosphorylation-Dependent Modulation of Survivin-Caspase-9 Interaction

A potential association of survivin with target caspase(s) at mitosiswas investigated. Immunoprecipitates of HA-survivin or HA-survivin(T34A) from viable HeLa cells 24 h-after transfection revealed thepresence of associated ˜46 kDa proform caspase-9, by Western blotting(FIG. 5A). In contrast, immunoprecipitates of wild type survivin orsurvivin (T34A) did not contain another upstream initiator caspase,caspase-8, by Western blotting (FIG. 5B). Co-immunoprecipitation andWestern blotting experiments were repeated from adherent ornon-adherent, i.e. mitotic/apoptotic, HeLa cells 48-h aftertransfection. As shown in FIG. 5C, HA-survivin immunoprecipitates fromadherent or non-adherent HeLa cell extracts revealed the presence ofassociated ˜35 kDa active caspase-9, by Western blotting (FIG. 5C).Analysis of the supernatants from these immunoprecipitates revealed apredominant 46 kDa proform caspase-9 band in extracts from adherentcells, and ˜35 kDa active caspase-9 in mitotic/apoptotic extracts (FIG.5C). In contrast, immunoprecipitates of non-phosphorylatable HA-survivin(T34A) from adherent, i.e. viable, HeLa cells contained barelydetectable amounts of ˜35 kDa active caspase-9, and no ˜46 kDa proformcaspase-9, by Western blotting (FIG. 5C). When similar experiments wererepeated using mitotic/apoptotic HeLa cell extracts, immunoprecipitatesof HA-survivin (T34A) did not contain associated preform or activecaspase-9 bands, by Western blotting (FIG. 5C). Consistent with the datapresented above, supernatants from these immunoprecipitates contained˜46 kDa proform caspase-9 in adherent cells, and ˜35 kDa activecaspase-9 in mitotic/apoptotic extracts (FIG. 5C). To determine if Thr³⁴phosphorylation affected the recognition of survivin for active/preformcaspase-9 at mitosis, co-immunoprecipitation and Western blottingexperiments were carried out from synchronized HeLa cells during cellcycle progression. Immunoprecipitates of wild type survivin or survivin(T34A) from interphase HeLa cells 0 or 6 h after thymidine releasecontained proform/active caspase-9 bands of ˜46 kDa and ˜35 kDa,respectively (FIG. 5D). However, in synchronized HeLa cells enteringmitosis 12 h after thymidine release, HA-survivin immunoprecipitatesbecame exclusively associated with ˜35 kDa active caspase-9, whereasboth proform and active caspase-9 bands were detected in the supernatant(FIG. 5D). In contrast, immunoprecipitates of non-phosphorylatablesurvivin (T34A) from mitotic HeLa cells did not contain associatedpreform or active caspase-9 bands, which accumulated in the relatedsupernatant (FIG. 5D). In control experiments, entry into mitosis ofsynchronized HeLa cells 12 h after thymidine release was demonstrated byDNA content analysis and flow cytometry (not shown).

B. Phosphorylation-Dependent Localization of Survivin-Caspase-9 Complex,In Vivo.

By immunofluorescence and confocal microscopy, HA-survivin andHA-survivin (T34A) transfected in HeLa cells bound to mitotic spindlemicrotubules at metaphase (not shown), and accumulated in midbodies attelophase, indistinguishably from endogenous survivin (FIG. 6A, B). InHA-survivin transfectants, simultaneous labeling for caspase-9 revealeda diffuse cytoplasmic reactivity and a prominent co-localization ofcaspase-9 with survivin in midbodies at different stages of telophase(FIG. 6A), in agreement with the co-immunoprecipitation data presentedabove. HeLa cells transfected with HA-survivin (T34A) also exhibited adiffuse labeling for caspase-9 throughout the cytoplasm (FIG. 6B).However, in the presence of non-phosphorylatable HA-survivin (T34A),caspase-9 selectively lost its localization to midbodies and did notco-associate with survivin (T34A) at telophase (FIG. 6B), consistentwith the dissociation of a survivin (T34A)-active caspase-9 complex (seeabove). In control experiments, another survivin point mutant in theBaculovirus IAP repeat (BIR), Leu⁶⁴→Ala, did not cause apoptosis (notshown), and physically co-localized with endogenous caspase-9 inmidbodies at telophase (FIG. 6C).

C. Caspase-9-Dependent Apoptotic Checkpoint at Mitosis.

To determine if active caspase-9 was responsible for apoptosis inducedby non-phosphorylatable survivin (T34A). By Western blotting, apoptosisof cell cycle synchronized Tet-F5.C4 cells was associated withtime-dependent cleavage of ˜46 kDa proform caspase-9 and generation of˜35 kDa and ˜37 kDa active caspase-9 bands (FIG. 7A). Consistent withthe kinetics of apoptosis of Tet-F5.C4 cells, the generation of activecaspase-9 bands began 9-h after thymidine release, increased at mitosis,and peaked in post-mitotic cells 24 h after release, with nearlycomplete disappearance of the ˜46 kDa preform caspase-9 band (FIG. 7A).Indistinguishable results of caspase-9 cleavage were observed in HeLacells treated with TNFα/cycloheximide (FIG. 7A). In contrast, Tet+F5.C4cells did not exhibit proteolytic cleavage of ˜46 kDa caspase-9 atmitosis (FIG. 7A). In other experiments, transfection of HeLa cells witha caspase-9 (C287A) dominant negative mutant (Duan et al., 1996)inhibited nuclear fragmentation and chromatin condensation induced byetoposide (FIG. 7B), in agreement with previous observations (Pan etal., 1998). Expression of caspase-9 (C287A) also reversed HeLa cellapoptosis induced by survivin (T34A) to background levels observed invector control transfectants (FIG. 7B), and restored the co-localizationof caspase-9 and endogenous survivin at midbodies (not shown). Incontrol experiments, co-transfection of HeLa cells with a caspase-8(C360S) dominant negative mutant (Boldin et al, 1996) did not reduceapoptosis induced by survivin (T34A), whereas it completely inhibitedcell death induced by TNFα/cycloheximide (FIG. 7B), in agreement withprevious observations (Boldin et al., 1996).

The results of Examples 1-3 show that survivin, a IAP apoptosisinhibitor (Deveraux and Reed, 1999) over-expressed in cancer (Ambrosiniet al., 1997; Velculescu et al., 1999), physically associates with thecyclin-dependent kinase complex, p34^(cdc2)-cyclin 131 (Nurse, 1994),and is phosphorylated by p34^(cdc2) on Thr³⁴. Based on the phenotype ofcells expressing a non-phosphorylatable survivin (T34A) mutant,phosphorylation on Thr³⁴ switches the affinity of survivin for activeversus proform caspase-9, and stabilizes this anti-apoptotic complex atmitosis (FIG. 13). Conversely, lack of survivin phosphorylation byp34^(cdc2) causes dissociation of the survivin-active caspase-9 complex,selective mislocalization of caspase-9 from midbodies, andcaspase-9-dependent apoptosis of cells traversing mitosis (FIG. 13).

Example 4 Method of Identifying an Agent that Modulates Phosphorylationof Survivin

The interactions described above between survivin and p34^(cdc2)-cyclinB1 kinase complex allow for the development of assays to identify anagent which modulates the phosphorylation of survivin. Such assays use,as common steps, a step of contacting survivin and p34²-cyclin B1 kinasecomplex in the presence of the agent and a step of determining whetherthe agent modulates the phosphorylation of survivin by the kinasecomplex. Any means to quantitate phosphorylation of survivin may beused.

In one format, the ability of an agent to modulate the phosphorylationof survivin is assayed. Baculovirus-expressed p34^(cdc2)-cyclin B1kinase complexes are incubated with 1 μg histone H1, 6 μg of wild typesurvivin in the presence and absence of an agent in a 25 μl reactionmixture containing 20 mM HEPES, pH 7.4, 10 mM MgCl₂, 0.5 mM DTT, 10 μMATP plus 10 μCi of ³²P-ATP (Amersham) for 30 min at 30° C. The tworeactions are terminated by addition of 25 μl of 2× Laemmli samplebuffer. To quantitate the amount of phosphorylated survivin, the abovesamples are electrophoresed on a 5-20% SDS polyacrylamide gel, andphosphorylated bands are visualized by autoradiography. Equal proteinloading in each kinase reaction is confirmed by Coomassie blue stainingof the gel. The ability of the agent to modulate phosphorylation ofsurvivin by p34^(cdc2)-cyclin B1 kinase complex is then determined bycomparing the amount or quantity of phosphorylated survivin in thesamples exposed to the agent and the amount or quantity ofphosphorylated survivin in the control (non-agent exposed) sample. Theamount of phosphorylated survivin is quantitated by cutting the survivingel bands out and counted in a scintillation counter. The amount ofphosphorylated survivin could also be determined by quantitativedensitometry.

Example 5 Methods of Identifying an Agent that ModulatesSurvivin/p34^(cdc2)-Cyclin B1 Kinase Complex Association orPhosphorylated Survivin/Caspase-9 Complex Association

The interactions described above between survivin and p34^(cdc2)-cyclinB1 kinase complex and between phosphorylated survivin and caspase-9allow for the development of assays to identify an agent which modulatessurvivin and p34^(cdc2)-cyclin B1 kinase complex association andsurvivin and caspase-9 association. Such assays may use, as commonsteps, a step of contacting survivin and p34^(cdc2)-cyclin B1 kinasecomplex in the presence of the agent and a step of determining whetherthe agent modulates the binding of survivin with the kinase complex orthe binding of survivin with caspase-9.

In one format, the ability of an agent to modulatesurvivin/p34^(cdc2)-cyclin B1 kinase complex association is assayed.Asynchronously growing HeLa cells are transfected with the nucleic acidencoding survivin and detergent-solubilized after 24 h. One sample ofdetergent solubilized cells is incubated with a test agent. The secondsample is not incubated with any test agent. The samples areimmunoprecipitated with an antibody to p34^(cdc2), separated byelectrophoresis, transferred to nylon membranes and immunoblotted withp34^(cdc2) antibodies. The ability of the agent to modulatesurvivin/p34^(cdc2)-cyclin B1 kinase complex interaction is thendetermined by comparing the positions of p34^(cdc2)-cyclin B1 kinasecomplex on the gel in the presence and absence of the agent.

Alternatively, agents that modulate survivin/p34^(cdc2)-cyclin B1 kinasemay be identified by using an in vitro kinase assay, similar to the onedescribed under Example 4. Determining the amount of phosphorylatedsurvivin, by for example, use of the phosphorylated survivin specificantibody described above, and comparing the amount of phosphorylatedsurvivin in the presence and absence of the test agent enables theskilled artisan to determine whether the agent inhibits thephosphorylation of survivin and/or an interaction between survivin andp34^(cdc2)-cyclin B1 kinase.

In another format, the ability of an agent to modulate phosphorylatedsurvivin/caspase-9 association is assayed. Baculovirus-expressedp34^(cdc2)-cyclic, B1 kinase complexes are incubated with 1 μg historicH1, 6 μg of wild type survivin in a 25 μl reaction mixture containing 20mM HEPES, pH 7.4, 10 mM MgCl₂, 0.5 marl DTT, 10 μM ATP plus 10 μCi of³²P-ATP (Amersham) for 30 min at 30° C. The reaction is terminated.Caspase-9 and a test agent is added to the sample. In the control samplecontaining baculovirus-expressed p34^(cdc2)-cyclin B1 and survivin, onlycaspase-9 is added. The association between phosphorylated survivin andcaspase-9 is then detected. For instance, the samples areimmunoprecipitated with an antibody to caspase-9, separated byelectrophores erred to nylon membranes and immunoblotted with caspase-9antibodies. The ability of the agent to modulate survivin/caspase-9interaction is then determined by comparing the positions of caspase-9on the gel in the presence and absence of the agent.

Example 6 The Effect of Survivin Antagonists on Human Melanoma Cells

YUSAC-2 human melanoma cells constitutively expressing endogenoussurvivin (Grossman et al., 1999b) were stably-transfected with survivinantagonists under the control of a tetracycline (tet)-regulated(“tet-off”) promoter system (Shockett et al, 1995).

The Thr³⁴→Ala mutation was introduced by site-directed mutagenesis intothe 1.6 kb human survivin cDNA (Ambrosini et al., 1997) using theoligonucleotide 5′-GGCTGCGCCTGCgCCCCGGAGCGGATG-3′ (SEQ ID NO: 2) and theGeneEditor system (Promega, The Thr³⁴→Ala mutation was introduced bysite-directed mutagenesis into the 1.6 kb human survivin cDNA (Ambrosiniet al., 1997) using the oligonucleotide5)-GGCTGCGCCTGCgCCCCGGAGCGGATG-3′ (SEQ ID NO: 3) and the GeneEditorsystem (Promega, Madison, Wis.) according to the manufacturer'sinstructions. This mutant was evaluated

along with other survivin BIR domain mutants for dominant-negativefunction by the ability to induce spontaneous apoptosis upon transienttransfection into Hela cells, as described (Li et al., 1998), and foundto be particularly effective. The survivin cDNA containing the Thr³⁴→Alamutation was cloned into the HindIII-SpeI sites of pTet-splice(Stratagene, La Jolla, Calif.). To generate the antisense construct, thewild-type survivin cDNA was cloned into pTet-splice in the reverseorientation.

These two survivin antagonists were cloned into pTet-splice downstreamof the regulatory sequences of the tet-resistance operon (TetO). Theplasmid pTA-Neo, containing the tet-controlled transactivator (tTA)sequence downstream of TetO, and a neomycin resistance gene, was kindlyprovided by David Schatz (Yale University School of Medicine). In thistandem plasmid system, tet prevents tTA binding to TetO andtranscription of the transgene; in the absence of tet, tTA upregulatesits own transcription and the transgene is expressed.

YUSAC-2 cells (Grossman et al., 1999) were transfected in 6-well platesby the simultaneous addition of 0.8 μg antagonist vector, 0.8 μgpTA-Neo, 0.5 μg tet (Sigma, St. Louis, Mo.), and 5 μl Lipofectamine(Life Technologies, Gaithersburg, Md.) per well. After 9 hours, thetransfection medium was aspirated and replaced with normal mediumcontaining 0.5 μg/ml tet. Forty-eight hours from the start oftransfection, cells were trypsinized, washed, and replated at lowdensity in 15×150 mm plates in medium containing 1.5 mg/ml G418 (LifeTechnologies), 2 mM sodium hydroxide and 0.5 μg/ml tet. This selectionmedium was changed every six days, and after three weeks 96 colonieswere transferred to U-bottom microtiter wells for expansion andscreening on the basis of differential growth in the presence andabsence of tet. Of the BIR mutant-transfected clones, three did not growin the absence of tet and two of them (designated F5.C4 and F5.E5) wererecloned by limiting dilution. Of the antisense-transfected clones, onlyone (designated B8) exhibited tet-dependent growth. Cells weremaintained in selection medium containing G418 and tet.

Upon withdrawal of tet from the culture medium, YUSAC-2 subclone F5.C4,transfected with the Thr³⁴→Ala mutant, strongly expressed a 16.5 kDainduced survivin band by Western blotting (FIG. 8A). By contrast, nomodulation of survivin expression was observed when tet was present inthe culture medium (FIG. 5A). Another subclone transfected with theThr³⁴→Ala mutant, F5.E5, similarly demonstrated tet-regulated transgeneexpression (not shown). In subclone B8, transfected with survivinantisense, removal of tet from the medium was associated with rapidexpression of survivin antisense RNA as assessed by RT-PCR (not shown).Tet-regulated expression of the Thr³⁴→Ala mutant or survivin antisenseresulted in a progressive time-dependent loss of mitotic (G2/M) cellsand coincident accumulation of apoptotic cells with hypodiploid (sub-G1)DNA content, as assessed by propidium iodide staining and flow cytometry(FIG. 8B, and not shown). Tet-deprived F5.C4 cells exhibited apoptoticnuclear morphology and stained intensely for internucleosomal DNAfragmentation by TUNEL (FIG. 8C). By contrast, F5.C4 cells cultured inthe presence of tet demonstrated normal mitotic progression (FIG. 8B)and lack of TUNEL reactivity (FIG. 8C).

Example 7 The Effect of Survivin Antagonists on CB.17 ImmunodeficientMice

To determine whether interference with survivin function by regulatedexpression of survivin antagonists could block melanoma tumor formationin CB.17 immunodeficient mice, mice were monitored for 8 weeks followingsubcutaneous injection, and tumor measurements taken at 4- and 8-weektime points (Table I). It was determined that YUSAC-2 cells consistentlyform localized tumors in 6 to 8-week-old female CB.17 SCID/beige mice(Taconic Farms, Germantown, N.Y.) approximately 3-4 weeks followingsubcutaneous injection of 1-3×10₆ cells. Animals were monitored for upto 4 months, and neither mortality nor gross metastasis is associatedwith increasing tumor size (up to 5000 mm ₃) or ulceration. One dayprior to injection, mice were shaved on the right flank, and the regulardrinking water was replaced with 5% sucrose alone or containing 100mg/ml tet as described (Shockett et al., 1995). Cells were harvested inlog-phase growth, washed twice in PBS, resuspended in PBS (12×10₆ cellsper ml) and injected (0.25 ml, 3×10₆ cells) subcutaneously. The drinkingwater was changed every 2-3 days. Tumor size was determined by theproduct of two perpendicular diameters and the height above the skinsurface.

Untransfected YUSAC-2 cells readily formed localized nodular amelanotictumors, and tumor formation was not affected by the addition or absenceof tet from the drinking water (FIG. 8D). Histologic analysis revealedsheets of large epitheloid malignant cells that stained positively forHMB-45, a marker of human melanoma cells (not shown; Kikuchi et al.,1996). The HMB-45 antibody (Zymed Laboratories, San Francisco, Calif.)was used according to the manufacturer's instructions. By contrast,F5.C4 cells transfected with survivin Thr³⁴→Ala did not form tumors in13 of 15 (87%) animals when tet was withheld from the drinking water(FIG. 8D, Table I). These animals remained tumor-free for an additional3-month observation period. Tumors that formed in two tet-deprivedanimals were considerably smaller in size and appeared with a markedlydelayed onset compared to those in animals given tet (Table I). Similarresults were obtained in animals injected with subclones F5.E5 and B8,transfected with survivin Thr³⁴→Ala and survivin antisense, respectively(Table I).

Table 1. Summary of tumor formation for all animals. CB.17 mice wereinjected with cells and tet was added or withheld from the drinkingwater. In this “tet-off” promoter system, the transgene is expressedonly in the absence of tet. Tumor incidence is reported for animalsobserved at 4 and 8 weeks as indicated. Mean tumor size (mm³)±standarddeviation is noted only for mice that had formed tumors at the indicatedobservation points. The slightly smaller size of tumors formed bysurvivin antagonist subclones compared to untransfected YUSAC-2 cellsmay be due to some leakiness of the promoter system or an unexplainedconsequence of transgene insertion.

4 Weeks 8 Weeks Cell line Transgene Tet Tumors formed Tumor size Tumorsformed Tumor size YUSAC-2 None present 5/5 (100%) 295 ± 129 5/5 (100%)2580 ± 1426 YUSAC-2 None absent 5/5 (100%) 358 ± 283 5/5 (100%) 2086 ±1228 F5.C4 Survivin Thr³⁴→Ala present 3/4 (75%)  181 ± 18  4/4 (100%)1533 ± 1019 F5.C4 Survivin Thr³⁴→Ala absent 0/15 (0%)   — 2/15 (13%) 278 ± 116 F5.E5 Survivin Thr³⁴→Ala present 3/4 (75%)  132 ± 115 3/4(75%)  924 ± 710 F5.E5 Survivin Thr³⁴→Ala absent 0/5 (100%) — 0/5 (100%)— B8 Survivin antisense present 5/5 (100%) 153 ± 98  5/5 (100%) 1249 ±559  B8 Survivin antisense absent 0/5 (100%) — 2/5 (40%)  164 ± 23 

Example 8 The Effect of Survivin Antagonists on Established Tumors

Next, the effect of survivin Thr³⁴→Ala on established tumors wasassessed. Fifteen mice were injected with F5.C4 cells and tet wasprovided to permit tumor formation. Once tumors became apparent (20-50mm³), tet was withheld from the drinking water and tumors were monitoredfor 5 weeks. None of the tumors grew at a rate comparable to F5.C4tumors in animals maintained on tet (FIG. 10A). Rather, a spectrum ofgrowth curves with some tumors failing to grow at all after tet waswithdrawn was observed (FIG. 10A). Histologic analysis revealed thatinduction of survivin Thr³⁴→Ala was associated with massive areas ofnecrosis and prominent cellular apoptosis by TUNEL, suggesting thatchanges in tumor mass in these animals did not reflect actual tumorgrowth. Withdrawal of tet from F5.C4 tumors was also associated withlack of normal mitotic figures and the presence of aberrant mitoses,consistent with the dysregulation of mitotic progression associated withsurvivin targeting in vitro (Li et al., 1998). In the two small F5.C4tumors that formed in the absence of tet (Table I), a similar pattern ofapoptotic cell death was seen (not shown).

Next, it was determined whether these effects on tumor growth andviability observed in vivo could be attributed to spontaneous apoptosisinduced by tet-regulated expression of survivin Thr³⁴→Ala. Cell lineswere re-established from several of these tumors and assessed fortet-regulated induction of apoptosis in vitro. Tumors were surgicallyexcised and skin and subcutaneous tissues were dissected away. Tumorswere then cut into small pieces with a razor blade and dissociated intoa single-cell suspension by vigorous vortexing in PBS. Cells were washedtwice in PBS, resuspended in selection medium containing G418 and tet,and cultured for 2-3 passages. These cells retained tet-responsivenessas removal of tet from the culture medium was associated with generationof hypodiploid (apoptotic) cells by DNA content analysis (FIG. 10B). Thepersistence of some viable cells in tet-deprived tumors may reflect aninability to remove tet completely from the animal and achieve maximaltransgene expression in vivo. In addition, inhibition of survivin maynot eliminate non-dividing cells given its selective action during theG2/M phase of the cell cycle (Li et al., 1999).

Although the present invention has been described in detail withreference to examples above, it is understood that various modificationscan be made without departing from the spirit of the invention.Accordingly, the invention is limited only by the following claims.

ADDITIONAL REFERENCES

The following articles are hereby incorporated by reference in theirentirety:

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1-29. (canceled)
 30. A method of inhibiting the growth of a tumor cellcomprising administering an effective amount of an antisense survivinnucleic acid to the tumor cell.
 31. The method of claim 30, wherein thetumor cell is in a tumor mass.
 32. The method of claim 31, wherein thetumor mass is present in a mammal.
 33. The method of claim 32, whereinthe tumor mass is a melanoma.
 34. The method of claim 31, wherein themethod inhibits the growth of a tumor mass in vivo.
 35. The method ofclaim 30, wherein the method inhibits the growth of a tumor cell invitro.
 36. The method of claim 30, wherein the method inhibits thegrowth of a tumor cell is in vivo.